tag:blogger.com,1999:blog-15741403324075919672024-03-12T15:37:19.988-07:00Spirochetes UnwoundBlogging about those twisty bacteria known as spirochetesMicrobe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.comBlogger78125tag:blogger.com,1999:blog-1574140332407591967.post-79455158550687609192016-12-31T22:20:00.001-08:002016-12-31T23:19:52.911-08:00Antibiotic cream is NOT 100% effective in preventing Lyme diseaseA topical antibiotic cream applied to tick bites did not perform any better than placebo in preventing Lyme disease, according to results of a randomized clinical trial conducted in Europe. The study was published in <i>Lancet Infectious Diseases</i>.<br />
<br />
I wasn't planning to blog about the study, but I changed my mind after a reader emailed me a link to a news article reporting that the antibiotic cream was 100% effective. The lead investigator even claimed, "None of the test subjects went on to develop Lyme borreliosis." As described by the <a href="https://www.yahoo.com/news/antibiotic-cream-could-prevent-lyme-disease-research-171634711.html?ref=gs" target="_blank">news sources</a>, seven subjects in the control group developed Lyme disease. But the <a href="http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(16)30529-1/abstract" target="_blank">abstract</a> of the paper states clearly that the antibiotic cream (azithromycin being the antibiotic) was not any better than the control cream; the investigators were even told to stop recruiting additional patients because the results was so clear with the patients who had already completed the study:<br />
<br />
<blockquote>
The trial was stopped early because an improvement in the primary endpoint in the group receiving azithromycin was not reached. At 8 weeks, 11 (2%) of 505 in the azithromycin group and 11 (2%) of 490 in the placebo group had treatment failure.</blockquote>
<br />
So how is it possible for the lead author to claim that "none" of the subjects treated with azithromycin came down with Lyme disease? The answer lies with the very last sentence of the abstract:<br />
<br />
<blockquote>
A subgroup analysis in this study suggested that topical azithromycin reduces erythema migrans after bites of infected ticks.</blockquote>
<br />
The subgroup analysis was done post-hoc (after looking at the data). I won't dwell on why we shouldn't make definitive conclusions from any post-hoc analysis since the investigators themselves emphasized its exploratory nature in the Discussion of their paper. However, even if you set that aside, you'll find another problem with the post-hoc analysis if you dig into the numbers. <br />
<br />
Before I tell you what the problem is, let me first describe the study in greater detail so that you understand the issues that led to the post-hoc analysis.<br />
<br />
The subjects were adults who had been bitten by a tick within the previous 72 hours and were able to save the tick. The subjects were randomized to receive a topical azithromycin cream or placebo cream. The cream was applied over the tick bite twice a day for three straight days. The patients were followed for 8 weeks. They were monitored for erythema migrans (EM), the characteristic rash of Lyme disease. Blood was drawn for serological testing at the beginning and at the end of the 8 week study period. "Treatment failure" was defined as the appearance of EM, seroconversion, or both by the end of 8 weeks. The ticks were tested for the bacteria that cause Lyme disease (<i>Borrelia garinii</i>, <i>B. afzelii</i>, and <i>B. burgdorferi</i>) by PCR.<br />
<br />
As I alluded to earlier, the independent committee monitoring the trial recommended that the investigators stop recruiting new subjects. Among the patients who already completed the study, the group receiving azithromycin did not fare any better than the placebo group, and recruiting more patients to the study was unlikely to change the conclusion. I provided the numbers above, but you can also find them in the table below ("ITT population," first row of data).<br />
<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="https://4.bp.blogspot.com/-3-1-BD6sUao/WGiUERSFWBI/AAAAAAAAAs4/mLILzuWHAhUr13BLYbTI0OJhHT96Fx_wgCLcB/s1600/Schwameis16-t2.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="288" src="https://4.bp.blogspot.com/-3-1-BD6sUao/WGiUERSFWBI/AAAAAAAAAs4/mLILzuWHAhUr13BLYbTI0OJhHT96Fx_wgCLcB/s400/Schwameis16-t2.png" width="400" /></a></div>
<br />
The researchers also did a pre-planned subanalysis with the per-protocol group, an idealized situation to directly test the question, "Does topical azithromycin prevent Lyme disease in those who are bitten by an <i>infected</i> tick?". Patients bitten by a PCR-negative tick were excluded from the subanalysis. The small number of patients who failed to follow or complete the study protocol were also excluded.<br />
<br />
Again, azithromycin was not any better than placebo in preventing EM or seroconversion (see table, "Per-protocol population"). Treatment failure was observed in 5% (3/62) of the azithromycin group and 7% (5/72) of the placebo group (P = 0.34).<br />
<br />
The researchers could have stopped the analysis there and write up the study, but the monitoring committee pointed out that none of the patients in the azithromycin group had erythema migrans by day 30 whereas five in the placebo group did. The committee suggested that the investigators do a post-hoc subgroup analysis using a modified definition of treatment failure as EM by 30 days. Seroconversion was removed from the modified definition.<br />
<br />
Looking at the numbers in the table ("Reanalyzed ITT population"), we now see where the news media got their numbers. No one in the azithromycin group (0/87, 0%) had EM by day 30, but seven in the placebo group (7/87, 8%) did. The difference was statiscially significant (absolute risk reduction in those receiving azithromycin: 8.05%, 95% CI 1.18-14.91). So, it's true that azithromycin prevented Lyme disease in all who were bitten by an infected tick - but only if you ignored the two patients who came down with EM after day 30 and a third patient who seroconverted.<br />
<br />
This is why I'm so baffled by the lead author's quote, which I will repeat: "None of the test subjects went on to develop Lyme borreiosis." I'm guessing that the two patients with delayed EM would disagree.<br />
<br />
<br />
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Lancet.+Infectious+Diseases&rft_id=info%3Apmid%2F28007428&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Topical+azithromycin+for+the+prevention+of+Lyme+borreliosis%3A+a+randomised%2C+placebo-controlled%2C+phase+3+efficacy+trial.&rft.issn=1473-3099&rft.date=2016&rft.volume=&rft.issue=&rft.spage=&rft.epage=&rft.artnum=&rft.au=Schwameis+M&rft.au=K%C3%BCndig+T&rft.au=Huber+G&rft.au=von+Bidder+L&rft.au=Meinel+L&rft.au=Weisser+R&rft.au=Aberer+E&rft.au=H%C3%A4rter+G&rft.au=Weinke+T&rft.au=Jelinek+T&rft.au=F%C3%A4tkenheuer+G&rft.au=Wollina+U&rft.au=Burchard+GD&rft.au=Aschoff+R&rft.au=Nischik+R&rft.au=Sattler+G&rft.au=Popp+G&rft.au=Lotte+W&rft.au=Wiechert+D&rft.au=Eder+G&rft.au=Maus+O&rft.au=Staubach-Renz+P&rft.au=Gr%C3%A4fe+A&rft.au=Geigenberger+V&rft.au=Naudts+I&rft.au=Sebastian+M&rft.au=Reider+N&rft.au=Weber+R&rft.au=Heckmann+M&rft.au=Reisinger+EC&rft.au=Klein+G&rft.au=Wantzen+J&rft.au=Jilma+B&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology">Schwameis M, Kündig T, Huber G, von Bidder L, Meinel L, Weisser R, Aberer E, Härter G, Weinke T, Jelinek T, Fätkenheuer G, Wollina U, Burchard GD, Aschoff R, Nischik R, Sattler G, Popp G, Lotte W, Wiechert D, Eder G, Maus O, Staubach-Renz P, Gräfe A, Geigenberger V, Naudts I, Sebastian M, Reider N, Weber R, Heckmann M, Reisinger EC, Klein G, Wantzen J, & Jilma B (2016). Topical azithromycin for the prevention of Lyme borreliosis: a randomised, placebo-controlled, phase 3 efficacy trial. <span style="font-style: italic;">The Lancet. Infectious Diseases</span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/28007428" rev="review">28007428</a></span><br />
<br />
<br />
<b><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Lancet.+Infectious+Diseases&rft_id=info%3Apmid%2F28007428&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Topical+azithromycin+for+the+prevention+of+Lyme+borreliosis%3A+a+randomised%2C+placebo-controlled%2C+phase+3+efficacy+trial.&rft.issn=1473-3099&rft.date=2016&rft.volume=&rft.issue=&rft.spage=&rft.epage=&rft.artnum=&rft.au=Schwameis+M&rft.au=K%C3%BCndig+T&rft.au=Huber+G&rft.au=von+Bidder+L&rft.au=Meinel+L&rft.au=Weisser+R&rft.au=Aberer+E&rft.au=H%C3%A4rter+G&rft.au=Weinke+T&rft.au=Jelinek+T&rft.au=F%C3%A4tkenheuer+G&rft.au=Wollina+U&rft.au=Burchard+GD&rft.au=Aschoff+R&rft.au=Nischik+R&rft.au=Sattler+G&rft.au=Popp+G&rft.au=Lotte+W&rft.au=Wiechert+D&rft.au=Eder+G&rft.au=Maus+O&rft.au=Staubach-Renz+P&rft.au=Gr%C3%A4fe+A&rft.au=Geigenberger+V&rft.au=Naudts+I&rft.au=Sebastian+M&rft.au=Reider+N&rft.au=Weber+R&rft.au=Heckmann+M&rft.au=Reisinger+EC&rft.au=Klein+G&rft.au=Wantzen+J&rft.au=Jilma+B&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology">Related posts</span></b><br />
<br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2011/09/flawed-study-claiming-prevention-of.html" target="_blank">A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2011/10/tale-of-two-more-studies-topical.html" target="_blank">A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease</a></li>
</ul>
Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-42762446710267674392016-12-23T20:17:00.001-08:002016-12-23T20:28:24.984-08:00The Lyme disease spirochete lives without thiamineThiamine, or vitamin B1, is vital for the survival of all living things. One of the biologically functional forms of thiamine, thiamine pyrophosphate (TPP), is essential for the catalytic activity of several critical metabolic enzymes. For this reason, we must get thiamine from the food that we eat (or the vitamin pills that we swallow). Microbes obtain the vitamin from their surroundings, but many can also make their own thiamine if it's not available.<br />
<br />
It turns out that the Lyme disease spirochete <i>Borrelia burgdorferi</i> does not need thiamine, as described by <a href="http://dx.doi.org/10.1038/NMICROBIOL.2016.213" target="_blank">Zhang and colleagues</a> in <cite>Nature Microbiology</cite>. The <i>B. burgdorferi</i> genome lacks the genes encoding the dedicated transporters that bring thiamine into the cell. The genes encoding the enzymes that produce thiamine are also absent. Chemical analysis of <i>B. burgdorferi</i> by HPLC failed to detect thiamine or TPP. Despite lacking the means to make or acquire thiamine, <i>B. burgdorferi</i> grew just fine in culture medium devoid of thiamine.<br />
<br />
The researchers conducted stringent tests to verify that <i>B. burgdorferi</i> could live without thiamine. To remove all traces of thiamine, they introduced the <i>bcmE</i> gene from <i>Clostridium botulinum</i> into the spirochete. The <i>bcmE</i> gene encodes an enzyme that rapidly breaks down thiamine. In culture, the spirochete grew at the same rate whether or not it had <i>bcmE</i>. The <i>bcmE</i> gene did not affect <i>B. burgdorferi's</i> ability to infect mice or to survive in feeding <i>Ixodes scapularis</i> ticks. The results of these experiments provided strong evidence that <i>B. burgdorferi</i> doesn't need thiamine to infect the tick vector or mouse.<br />
<br />
How does <i>B. burgdorferi</i> manage to live without thiamine? It can do without most of the enzymes that require the TPP coenzyme, but it's less obvious how <i>B. burgdorferi</i> copes without pyruvate dehydrogenase (PDH), a TPP-dependent enzyme that converts pyruvate to acetyl-CoA (see figure). Acetyl-CoA is an essential precursor to the bacterial cell wall, something that <i>B. burgdorferi</i> obviously needs. The researchers proposed that <i>B. burgdorferi</i> makes acetyl-CoA by an alternative pathway that starts with acetate. <i> B. burgdorferi</i> possesses the enzymes acetate kinase (ACK) and phosphate acetyltransferase (PTA), which convert acetate to acetyl-CoA (see figure).<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://3.bp.blogspot.com/-DMmtRSrHZ8s/WFyAYBMjM8I/AAAAAAAAAsc/QweAaBrlsaYoNVjUCkyMBH1yWO9LGP0xwCLcB/s1600/Zhang16-f4%2528sm%2529.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://3.bp.blogspot.com/-DMmtRSrHZ8s/WFyAYBMjM8I/AAAAAAAAAsc/QweAaBrlsaYoNVjUCkyMBH1yWO9LGP0xwCLcB/s1600/Zhang16-f4%2528sm%2529.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 from Zhang <i>et al</i>., 2016. Enzymes in red (PDC, PDH, and POX) require the TPP coenzyme. Metabolic pathways found in other bacteria but missing in <i>B. burgdorferi</i> are shown with dashed lines.</td></tr>
</tbody></table>
<br />
<i>B. burgdorferi</i> may not be alone in living without thiamine. The researchers also looked at the genomes of other bacterial pathogens that are transmitted by arthropods. <i>Borrelia hermsii</i> (relapsing fever), <i>Rickettsia prowazekii</i> (epidemic typhus), and <i>R. conorii</i> (Mediterranean spotted fever) were missing the genes for thiamine biosynthesis and the enzymes that use thiamine pyrophosphate as a coenzyme.<br />
<br />
The presence of the alternative pathway to acetyl-CoA synthesis assumes that acetate is available in the microenvironment surrounding the arthropod-borne pathogen. According to measurements presented in a <a href="http://dx.doi.org/10.1371/journal.ppat.1001104" target="_blank">2010 paper</a>, acetate is present in the midgut of fed <i>I. scapularis</i> ticks and in mouse blood. The <i>B. burgdorferi</i> protein BBA34 <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147597/" target="_blank">may be a transporter</a> that brings acetate into the cell.<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Nature+Microbiology&rft_id=info%3Apmid%2F27869793&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Lyme+disease+spirochaete+Borrelia+burgdorferi+does+not+require+thiamin.&rft.issn=&rft.date=2016&rft.volume=2&rft.issue=&rft.spage=16213&rft.epage=&rft.artnum=&rft.au=Zhang+K&rft.au=Bian+J&rft.au=Deng+Y&rft.au=Smith+A&rft.au=Nunez+RE&rft.au=Li+MB&rft.au=Pal+U&rft.au=Yu+AM&rft.au=Qiu+W&rft.au=Ealick+SE&rft.au=Li+C&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Zhang K, Bian J, Deng Y, Smith A, Nunez RE, Li MB, Pal U, Yu AM, Qiu W, Ealick SE, & Li C (2016). Lyme disease spirochaete Borrelia burgdorferi does not require thiamin. <span style="font-style: italic;">Nature Microbiology, 2</span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/27869793" rev="review">27869793</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+pathogens&rft_id=info%3Apmid%2F20862323&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Role+of+acetyl-phosphate+in+activation+of+the+Rrp2-RpoN-RpoS+pathway+in+Borrelia+burgdorferi.&rft.issn=1553-7366&rft.date=2010&rft.volume=6&rft.issue=9&rft.spage=&rft.epage=&rft.artnum=&rft.au=Xu+H&rft.au=Caimano+MJ&rft.au=Lin+T&rft.au=He+M&rft.au=Radolf+JD&rft.au=Norris+SJ&rft.au=Gherardini+F&rft.au=Wolfe+AJ&rft.au=Yang+XF&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Xu H, Caimano MJ, Lin T, He M, Radolf JD, Norris SJ, Gherardini F, Wolfe AJ, & Yang XF (2010). Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi. <span style="font-style: italic;">PLoS pathogens, 6</span> (9) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/20862323" rev="review">20862323</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Infection+and+immunity&rft_id=info%3Apmid%2F21628523&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Oligopeptide+permease+A5+modulates+vertebrate+host-specific+adaptation+of+Borrelia+burgdorferi.&rft.issn=0019-9567&rft.date=2011&rft.volume=79&rft.issue=8&rft.spage=3407&rft.epage=20&rft.artnum=&rft.au=Subba+Raju+BV&rft.au=Esteve-Gassent+MD&rft.au=Karna+SL&rft.au=Miller+CL&rft.au=Van+Laar+TA&rft.au=Seshu+J&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Subba Raju BV, Esteve-Gassent MD, Karna SL, Miller CL, Van Laar TA, & Seshu J (2011). Oligopeptide permease A5 modulates vertebrate host-specific adaptation of Borrelia burgdorferi. <span style="font-style: italic;">Infection and immunity, 79</span> (8), 3407-20 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21628523" rev="review">21628523</a></span><br />
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-23774530569743184512016-07-14T18:39:00.000-07:002016-07-14T18:39:58.639-07:00Are NETs involved in fighting Leptospira interrogans infections?Neutrophils are the most abundant white blood cells in the bloodstream. As the first immune cells to be recruited to infected tissues, they play a key role in the fighting microbial intruders. It's long been known that they engulf microbes by phagocytosis, which results in the microbes being imprisoned within phagosomes inside the neutrophil. Deadly proteases, antimicrobial proteins, and reactive oxygen species are released into the phagosome to kill the microbes.<br />
<br />
Another means used by neutrophils to kill microbes was <a href="http://www.ncbi.nlm.nih.gov/pubmed/15001782" target="_blank">discovered just a decade ago</a>. When mixed with bacteria, neutrophils cast nets of DNA impregnated with antimicrobial proteins to trap and kill the bacteria. The web-like DNA goes by the name "neutrophil extracellular trap" (NET). Several bacteria are known to trigger neutrophils to cast NETs, and NETs have even been observed by microscopy within infected tissues.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://4.bp.blogspot.com/-jFuFZuNedbA/V4e_FXDX0_I/AAAAAAAAArc/D2UwdyLzT4E5cBklT4HEnmkw0iN8QQg4QCLcB/s1600/Brinkmann04-f4h.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://4.bp.blogspot.com/-jFuFZuNedbA/V4e_FXDX0_I/AAAAAAAAArc/D2UwdyLzT4E5cBklT4HEnmkw0iN8QQg4QCLcB/s1600/Brinkmann04-f4h.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fluorescence staining of a neutrophil exudate in an appendicitis case. NETs are the fibrous material. Figure 4H from <a href="http://www.ncbi.nlm.nih.gov/pubmed/15001782" target="_blank">Brinkmann et al., 2004</a>. Bar = 50 μm. </td></tr>
</tbody></table>
A <a href="http://dx.doi.org/10.1371/journal.pntd.0003927" target="_blank">study published last year in <cite>PLOS NTD</cite></a> showed that the spirochete <i>Leptospira interrogans</i> is also killed by NETs. The image below shows the spirochetes trapped in a NET cast by a human neutrophil.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://1.bp.blogspot.com/-weYDAMzf4mc/V4fJKi-xntI/AAAAAAAAArs/R8hz39Xg8OUo47x0dtqL6rIvuQExX2G3QCLcB/s1600/Scharrig15-f1a.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://1.bp.blogspot.com/-weYDAMzf4mc/V4fJKi-xntI/AAAAAAAAArs/R8hz39Xg8OUo47x0dtqL6rIvuQExX2G3QCLcB/s1600/Scharrig15-f1a.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Human neutrophils were cultured with <i>L. interrogans</i> for 3 hours. <a href="http://dx.doi.org/10.1371/journal.pntd.0003927.g001" target="_blank">Figure 1A</a> from Scharrig <i>et al</i>., 2015. Bar = 50 μm.</td><td class="tr-caption" style="text-align: center;"><br /></td></tr>
</tbody></table>
The real question is whether NETs are involved in killing <i>L. interrogans</i>
during infection. To answer this question, the investigators turned to
the mouse model of leptospirosis. They found that the number of
spirochetes in the bloodstream more than doubled when the neutrophils in
the mice were depleted by injection of a monoclonal antibody targeting a
antigen located on the neutrophil surface. Later in the infection, there
was 10-fold more spirochetes in the kidneys of mice whose
neutrophils were depleted than in those with normal numbers of
neutrophils. This confirmed that neutrophils were involved in limiting
infections by <i>L. interrogans</i>, but did the neutrophils fight the infection by casting NETs?<br />
<br />
The investigators used an indirect method to measure
the amount of NETs generated during infection. Neutrophils often expel nuclear DNA in the form of <a href="https://en.wikipedia.org/wiki/Nucleosome" target="_blank">nucleosomes</a> to generate NETs. (Nucleosomes are assembled by wrapping nuclear DNA around <a href="https://en.wikipedia.org/wiki/Histone" target="_blank">histones</a>.) For this reason, the investigators measured the levels of free nucleosomes in the bloodstream of infected mice by ELISA. They concluded that NETs were generated by neutrophils in the bloodstream because they detected free nucleosomes in blood drawn from infected mice. Much less was detected when neutrophils were first depleted with the anti-neutrophil antibody, confirming that the main source of free nucleosomes was neutrophils.<br />
<br />
These results don't convince me that NETs are generated by neutrophils during <i>L. interrogans</i> infection. There could be other reasons for free nucleosomes being present in the bloodstream. For example, nucleosomes could be released from neutrophils simply dying from their battle against <i>L. interrogans</i>. More convincing evidence would be direct observation of NETs in infected animals, as done in <a href="http://www.ncbi.nlm.nih.gov/pubmed/22980329" target="_blank">this</a> study of mice with <i>E. coli</i> blood infections.<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+Neglected+Tropical+Diseases&rft_id=info%3Apmid%2F26161745&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Neutrophil+extracellular+traps+are+involved+in+the+innate+immune+response+to+infection+with+Leptospira.&rft.issn=1935-2727&rft.date=2015&rft.volume=9&rft.issue=7&rft.spage=&rft.epage=&rft.artnum=&rft.au=Scharrig+E&rft.au=Carestia+A&rft.au=Ferrer+MF&rft.au=C%C3%A9dola+M&rft.au=Pretre+G&rft.au=Drut+R&rft.au=Picardeau+M&rft.au=Schattner+M&rft.au=G%C3%B3mez+RM&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Scharrig E, Carestia A, Ferrer MF, Cédola M, Pretre G, Drut R, Picardeau M, Schattner M, & Gómez RM (2015). Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira. <span style="font-style: italic;">PLoS Neglected Tropical Diseases, 9</span> (7) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/26161745" rev="review">26161745</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Science+%28New+York%2C+N.Y.%29&rft_id=info%3Apmid%2F15001782&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Neutrophil+extracellular+traps+kill+bacteria.&rft.issn=0036-8075&rft.date=2004&rft.volume=303&rft.issue=5663&rft.spage=1532&rft.epage=5&rft.artnum=&rft.au=Brinkmann+V&rft.au=Reichard+U&rft.au=Goosmann+C&rft.au=Fauler+B&rft.au=Uhlemann+Y&rft.au=Weiss+DS&rft.au=Weinrauch+Y&rft.au=Zychlinsky+A&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, & Zychlinsky A (2004). Neutrophil extracellular traps kill bacteria. <span style="font-style: italic;">Science (New York, N.Y.), 303</span> (5663), 1532-5 PMID: <a rev="review" href="http://www.ncbi.nlm.nih.gov/pubmed/15001782">15001782</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-54306192972328532622016-06-14T00:01:00.001-07:002016-06-14T09:01:03.021-07:00Xenodiagnosis to detect Borrelia burgdorferi in humansWe've seen that <a href="http://spirochetesunwound.blogspot.com/2016/05/resurgence-of-borrelia-burgdorferi-in.html" target="_blank">live <i>Borrelia burgdorferi</i> persists</a> (in unculturable form) when infected mice are treated with antibiotics. What we don't know is whether they persist in humans with post-treatment Lyme disease syndrome (PTLDS), which refers to the lingering long-term symptoms experienced by a minority of Lyme disease patients who have been treated with the standard course of antibiotics.<br />
<br />
In theory, one could simply determine whether <i>B. burgdorferi</i> can be detected in bits of tissue or blood extracted from volunteers with post-treatment symptoms. This is what was done in the mouse studies that I described in my <a href="http://spirochetesunwound.blogspot.com/2016/05/resurgence-of-borrelia-burgdorferi-in.html" target="_blank">previous post</a>. It's easy to culture <i>B. burgdorferi</i> from untreated mice that have been infected for a long time. However, humans are not mice. Except in those with Lyme arthritis, the spirochete is hard to detect by culture or PCR in patients at later stages of Lyme disease, even in those who haven't taken antibiotics.<br />
<br />
In fact, three of the four randomized controlled retreatment trials that I keep on bringing up on this blog included attempts to detect <i>B. burgdorferi</i> in cerebral spinal fluid or blood of PTLDS patients by culture and PCR. No specimen was culture positive except for one, and none were PCR positive. The single positive culture turned out
to be a contaminant.<br />
<br />
The rest of the scientific literature is littered with claims that Lyme <i>Borrelia</i> can be detected by culture or PCR in blood, urine, or CSF of treated patients. However, critics have raised several concerns about these studies. For instance, alternative explanations for the findings such as contamination or reinfection weren't ruled out.<br />
<br />
With all of this as background, <a href="http://www.ncbi.nlm.nih.gov/pubmed/24523212" target="_blank">Marques and colleagues</a> decided to test a different approach – xenodiagnosis. For this procedure, uninfected ticks are deliberately placed on the skin and left for several days to give them time to take a blood meal. If there are any spirochetes in the skin nearby, they will move towards the feeding site because they are attracted to the tick's saliva. The spirochetes then get drawn into the tick's feeding tube along with the blood meal. The fed ticks are then removed and tested for the presence of <i>B. burgdorferi</i>. The sensitivity of xenodiagnosis can be enhanced by placing multiple ticks to increase the chance that at least one tick will drink blood containing <i>B. burgdorferi</i>. Xenodiagnosis is done routinely with mice in the research setting, and I <a href="http://spirochetesunwound.blogspot.com/2016/05/resurgence-of-borrelia-burgdorferi-in.html" target="_blank">mentioned in my previous post</a> that <i>B. burgdorferi</i> can be detected in antibiotic-treated mice by xenodiagnosis.<br />
<br />
The first thing to do was a pilot study to make sure that the procedure was safe for volunteers. 25 subjects who had been treated for Lyme disease took part in the study. 10 of the 25 had PTLDS. Ten healthy volunteers and one subject with untreated erythema migrans (EM), the skin rash of early-stage Lyme disease, were included in the study.<br />
<br />
As for the ticks, the investigators bred and maintained <i>Ixodes scapularis</i> in the laboratory. The ticks were carefully screened to make sure they were free of known infectious agents.<br />
<br />
25-30 ticks were placed on each volunteer and covered with a special dressing to keep them in place (see images below). The ticks were left alone for a week so that they could consume a blood meal. Some of the fed ticks were tested for the presence of <i>B. burgdorferi</i> DNA by standard PCR or by a more sensitive technique: isothermal amplification followed by PCR and mass spectrometry (IA/PCR/ESI-MS). The remaining ticks were cultured or were placed on immune-deficient mice to determine whether <i>B. burgdorferi</i>, if present, could be transmitted.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://2.bp.blogspot.com/-vPgMA5e0wR4/V1TAyKL_cpI/AAAAAAAAArE/UDFxJLSgSLY1v2_nOqjwN8qOV5auJIF-wCLcB/s1600/Marques14-f1%2528sm%2529.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="211" src="https://2.bp.blogspot.com/-vPgMA5e0wR4/V1TAyKL_cpI/AAAAAAAAArE/UDFxJLSgSLY1v2_nOqjwN8qOV5auJIF-wCLcB/s400/Marques14-f1%2528sm%2529.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 from Marques <i>et al.</i>, 2014. Left panel: ticks covered with a special dressing on forearm. Right panel: feeding ticks attached to forearm, dressing removed.</td></tr>
</tbody></table>
<br />
So did anyone test positive by xenodiagnosis? Yes. <i>B. burgdorferi</i> DNA was detected in two subjects. One was the subject with untreated EM. This subject served as sort of a positive control. I say "sort of" because antibiotic therapy was started at the same time that the ticks were placed on the EM lesion – it would not have been ethical to delay treatment while the ticks were feeding. <i>B. burgdorferi</i> DNA was detected in two of the ten ticks tested. The subject was tested by xenodiagnosis again seven months later, and all ten ticks that were tested were negative for <i>B. burgdorferi</i> DNA.<br />
<br />
The other positive test came from one of the PTLDS subjects. One of the five ticks that were tested was positive for <i>B. burgdorferi</i> DNA. The same subject tested positive by xenodiagnosis again 8 months later: one of three ticks tested positive for <i>B. burgdorferi</i> DNA by IA/PCR/ESI-MS.<br />
<br />
Of course DNA doesn't equal viability. The study didn't provide much evidence that the DNA detected in the single case of PTLDS came from spirochetes that were alive at the time that the ticks were placed. A skin biopsy taken from where the xenodiagnostic ticks were feeding was culture negative, as were the fed ticks themselves. The ticks also failed to transmit <i>B. burgdorferi</i> to immune-deficient mice, a process that probably requires live, motile spirochetes. To be fair, this was just a pilot study with the primary goal to assess the safety of xenodiagnosis. Nothing terrible happened to the volunteers, although half experienced mild itching at the feeding site. The investigators are <a href="https://clinicaltrials.gov/ct2/show/NCT02446626" target="_blank">recruiting additional subjects</a> for a larger study to determine whether positive test results by xenodiagnosis are associated with post-treatment symptoms.<br />
<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+Infectious+Diseases&rft_id=info%3Apmid%2F24523212&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Xenodiagnosis+to+detect+Borrelia+burgdorferi+infection%3A+a+first-in-human+study.&rft.issn=1058-4838&rft.date=2014&rft.volume=58&rft.issue=7&rft.spage=937&rft.epage=45&rft.artnum=&rft.au=Marques+A&rft.au=Telford+SR+3rd&rft.au=Turk+SP&rft.au=Chung+E&rft.au=Williams+C&rft.au=Dardick+K&rft.au=Krause+PJ&rft.au=Brandeburg+C&rft.au=Crowder+CD&rft.au=Carolan+HE&rft.au=Eshoo+MW&rft.au=Shaw+PA&rft.au=Hu+LT&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Marques A, Telford SR 3rd, Turk SP, Chung E, Williams C, Dardick K, Krause PJ, Brandeburg C, Crowder CD, Carolan HE, Eshoo MW, Shaw PA, & Hu LT (2014). Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. <span style="font-style: italic;">Clinical Infectious Diseases, 58</span> (7), 937-45 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24523212" rev="review">24523212</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+Infectious+Diseases&rft_id=info%3Apmid%2F24523213&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Xenodiagnosis+for+posttreatment+Lyme+disease+syndrome%3A+resolving+the+conundrum+or+adding+to+it%3F&rft.issn=1058-4838&rft.date=2014&rft.volume=58&rft.issue=7&rft.spage=946&rft.epage=8&rft.artnum=&rft.au=Bockenstedt+LK&rft.au=Radolf+JD&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Bockenstedt LK, & Radolf JD (2014). Xenodiagnosis for posttreatment Lyme disease syndrome: resolving the conundrum or adding to it? <span style="font-style: italic;">Clinical Infectious Diseases, 58</span> (7), 946-8 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24523213" rev="review">24523213</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Expert+Review+of+Anti-infective+Therapy&rft_id=info%3Apmid%2F25301228&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Is+there+a+place+for+xenodiagnosis+in+the+clinic%3F&rft.issn=1478-7210&rft.date=2014&rft.volume=12&rft.issue=11&rft.spage=1307&rft.epage=10&rft.artnum=&rft.au=Telford+SR+3rd&rft.au=Hu+LT&rft.au=Marques+A&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Telford SR 3rd, Hu LT, & Marques A (2014). Is there a place for xenodiagnosis in the clinic? <span style="font-style: italic;">Expert Review of Anti-infective Therapy, 12</span> (11), 1307-10 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/25301228" rev="review">25301228</a></span><br />
<br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2014/03/video-microscopy-of-ticks-acquiring.html" target="_blank">Video microscopy of ticks acquiring the Lyme disease spirochete from mice</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2016/05/resurgence-of-borrelia-burgdorferi-in.html" target="_blank">Resurgence of <i>Borrelia burgdorferi</i> in mice a year after antibiotic treatment</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com3tag:blogger.com,1999:blog-1574140332407591967.post-81006385870231137202016-05-14T21:57:00.000-07:002016-05-15T10:56:26.812-07:00Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatmentAs a follow up to my <a href="http://spirochetesunwound.blogspot.com/2016/04/long-term-antibiotics-for-those-with.html" target="_blank">previous post</a>, I would like to say something about several mouse studies from Stephen Barthold's group. These papers are often cited by those who believe that retreatment is needed in patients who continue to experience symptoms following treatment of Lyme disease with conventional antibiotic regimens. The assumption is that post-treatment symptoms stem from spirochetes surviving the initial antibiotic therapy.<br />
<br />
In the 2008 and 2010 studies (described in detail <a href="http://spirochetesunwound.blogspot.com/2009/01/chronic-lyme-disease-in-mice.html" target="_blank">here</a> and <a href="http://spirochetesunwound.blogspot.com/2010/03/tigecycline-fails-to-eradicate.html" target="_blank">here</a>), Barthold's group gave doxycycline, ceftriaxone, or tigecycline to mice with disseminated <i>Borrelia burgdorferi</i> infection. As expected, all tissues were culture negative up to three months following antibiotic therapy. Tissues from untreated mice were culture positive. However, <i>B. burgdorferi</i> DNA and mRNA were detected by PCR in up to half the treated mice, and microscopy revealed a few intact spirochetes in collagen-rich tissues from these mice. Ticks allowed to feed on the treated mice even transmitted the spirochetes to other mice (albeit immune deficient ones), where <i>B. burgdorferi</i> DNA was detected by PCR. Clearly, the spirochetes that survived antibiotic treatment were alive despite being unculturable. <br />
<br />
Although live spirochetes remained following antibiotic therapy, there was no evidence that they were capable of causing disease. Lyme disease is driven by inflammation, but no inflammatory response in the form of infiltrating immune cells were seen in tissues harboring the spirochetes. A <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708393/" target="_blank">critic of the work</a> also pointed out that the number of spirochetes declined during the 3 months following treatment, implying that any lingering spirochetes would eventually disappear. It seemed unlikely that a similar phenomenon was responsible for persisting symptoms following treatment of Lyme disease in human patients, who may suffer with disabling symptoms for years.<br />
<br />
In 2014 Barthold's group came out with another paper, which I'm discussing here for the first time. Again, mice with disseminated <i>B. burgdorferi</i> infections were treated with antibiotics, ceftriaxone in this case. But this time, the mice were left for up to a year before their tissues were examined for the presence of <i>B. burgdorferi</i>. Control mice were mock treated with saline and examined along with the treated mice.<br />
<br />
There weren't any surprises when tissues were tested by culture. Most of the control mice were culture positive at all time points (2, 4, 8, and 12 months) with both tissues tested, the urinary bladder and the skin where <i>B. burgdorferi</i> was inoculated to initiate infection. None of the treated mice were culture positive at either site at any time point.<br />
<br />
PCR testing for <i>B. burgdorferi</i> DNA was done with tissue obtained from six sites in the mice. Ticks allowed to feed on the mice were also tested for the presence <i>B. burgdorferi</i> DNA by PCR in a method called xenodiagnosis. All saline-treated mice were PCR positive in most tissues tested, and most tested positive by xenodiagnosis.<br />
<br />
The results with the mice treated with ceftriaxone are shown in the table below. Each row represents a single mouse. Note that each tissue homogenate was tested three times.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://3.bp.blogspot.com/-coIy1T_IoFc/VzKe_mhWuCI/AAAAAAAAAqs/hgRmURccHk0zlOO6gKzV3Gnau0jrC_iwQCLcB/s1600/Hodzic14-t2.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="480" src="https://3.bp.blogspot.com/-coIy1T_IoFc/VzKe_mhWuCI/AAAAAAAAAqs/hgRmURccHk0zlOO6gKzV3Gnau0jrC_iwQCLcB/s640/Hodzic14-t2.png" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Table 2 from Hodzic <i>et al.</i>, 2014. "Interval" = time after completion of treatment; "Inoc" = skin from inoculation site; "HB" = heart base; "VM" = ventricular muscle; "QM" = quadriceps muscle; "Tt" = tibiotarsus; "XenoDx" = xenodiagnostic ticks (# ticks testing positive/# ticks placed on mouse).</td></tr>
</tbody></table>
<br />
They saw something remarkable with the mice left for 12 months. Although few tissues were positive at earlier time points, most tissues extracted from mice a year after treatment tested positive. 6 of the 8 mice also tested positive by xenodiagnosis. So, instead of eventually disappearing, the spirochetes proliferated starting at some point after 8 months elapsed following treatment. This resurgence occurred even though the spirochetes remained unculturable. <br />
<br />
Barthold's group also looked for evidence of inflammation. Despite the resurgence of spirochetes, they did not see much evidence of inflammation by microscopy of the tissues 12 months following antibiotic treatment. However, the researchers pointed out that no conclusions can be drawn about the ability of the persisting spirochetes to cause disease since inflammation was minimal even in saline-treated mice, which harbored culturable spirochetes.<br />
<br />
The researchers next looked for molecular evidence of inflammation. They measured transcript levels of 18 cytokines in the base of the heart, heart muscle, quadriceps muscle, and leg joint 12 months after treatment with ceftriaxone or saline. The levels of cytokine transcripts in the two groups were compared to those in age-matched uninfected mice. Not surprisingly, saline-treated mice had what the authors deemed a "proinflammatory" cytokine profile, most likely due to their ongoing infection. Antibiotic-treated mice also had a proinflammatory cytokine profile, although it differed from that of the saline-treated mice. This observation is the first to suggest that the mice were responding to persisting spirochetes that survived antibiotic treatment.<br />
<br />
In conclusion, the evidence is convincing that <i>B. burgdorferi</i> persists <i>in mice</i>
for a long time after antibiotic treatment. They don't eventually disappear and may
even proliferate. Whether these unculturable spirochetes are capable of
generating an inflammatory condition necessary for disease is less
clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes.<br />
<br />
Barthold's group caution readers from applying the findings too broadly:<br />
<blockquote>
Because of the controversial nature of these findings, they should not be over-interpreted and certainly not translated directly into clinical management of human Lyme borreliosis.</blockquote>
<br />
So is there any relevance of these findings to post-treatment symptoms in humans? I will touch upon this issue in a future post.<br />
<br />
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLOS+One&rft_id=info%3Apmid%2F24466286&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Resurgence+of+persisting+non-cultivable+Borrelia+burgdorferi+following+antibiotic+treatment+in+mice.&rft.issn=&rft.date=2014&rft.volume=9&rft.issue=1&rft.spage=&rft.epage=&rft.artnum=&rft.au=Hodzic+E&rft.au=Imai+D&rft.au=Feng+S&rft.au=Barthold+SW&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Hodzic E, Imai D, Feng S, & Barthold SW (2014). Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. <span style="font-style: italic;">PLOS One, 9</span> (1) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24466286" rev="review">24466286</a></span><br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2009/01/chronic-lyme-disease-in-mice.html" target="_blank">Chronic Lyme disease in mice?</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2010/03/tigecycline-fails-to-eradicate.html" target="_blank">Tigecycline fails to eradicate persisting <i>Borrelia burgdorferi</i></a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2016/04/long-term-antibiotics-for-those-with.html" target="_blank">Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com17tag:blogger.com,1999:blog-1574140332407591967.post-72353201527038297282016-04-23T20:37:00.000-07:002016-05-14T22:43:10.894-07:00Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme diseaseA Lyme disease study published a few weeks ago in the <cite>New England Journal of Medicine</cite> has received a lot of coverage in the <a href="http://www.npr.org/sections/health-shots/2016/03/30/472411123/study-prolonged-antibiotic-treatment-gave-no-relief-for-lasting-lyme-symptoms" target="_blank">press</a>. According to the <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=27028911[uid]" target="_blank">abstract</a> of the study, Berende and colleagues conducted a randomized placebo-controlled clinical trial to test the effectiveness of <strike>long-term</strike> "longer-term" antibiotics in treating <strike>"longer-term"</strike> chronic symptoms "attributed" to Lyme disease.<br />
<br />
As many readers of this blog know, treatment of Lyme disease is a controversial topic. Antibiotics are effective in treating Lyme disease, but
10-20% experience symptoms such as fatigue, muscular aches,
and joint pain for at least 6 months following conventional treatment. The cause of the persisting symptoms is not known. They could be
due to tissue damage caused by the infection, ongoing inflammation, or bacteria that survived antibiotic treatment. Mainstream medical societies such as the <a href="http://www.idsociety.org/About_IDSA/" target="_blank">IDSA</a> do not believe that lingering infection is responsible for the persisting symptoms, and they do not recommend retreatment with antibiotics. Four randomized controlled studies conducted in the U.S. showed little benefit of
retreating these patients with antibiotics for up to 3
months. On the other hand, not-so-mainstream groups such as <a href="http://www.ilads.org/" target="_blank">ILADS</a>
dispute the interpretation of the data. They insist that the treatment
groups did show some improvement and that longer
treatment regimens lasting longer than 3 months are needed for complete recovery of these patients.<br />
<br />
There is another group of patients that also suffer from enduring fatigue, muscle aches, and joint pain. They may or may not have had Lyme disease in the past, but their ongoing symptoms stem from some other condition. Unfortunately, they may be misdiagnosed with "chronic Lyme disease" and end up being treated for a long time with antibiotics in an attempt to eradicate an infection that they don't have.<br />
<br />
The new NEJM paper describes a randomized placebo-controlled trial that
was conducted in the Netherlands. 281 subjects who had been experiencing chronic symptoms blamed on Lyme disease (fatigue, muscle aches, joint pain) were randomized into three groups. All three
groups were treated with ceftriaxone intravenously for two weeks. The subjects were next given oral antibiotics or placebo for 12 weeks.
One group was treated with doxycycline, another group with both
clarithromycin and hydroxychloroquine, and the third group was given
placebo pills.<br />
<br />
The graph below shows that the physical quality of life, the primary outcome measure, improved a little for all groups. Because there was no difference in outcome among the three groups, the authors concluded that longer-term antibiotics were no better than short-term antibiotics in alleviating symptoms. We don't know whether the initial two-week treatment with ceftriaxone had anything to do with the slight
improvement since there was no true placebo (antibiotic-free) group.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://3.bp.blogspot.com/-B3zrWIjfx-8/VxQ92ovg0wI/AAAAAAAAAqU/Wu-R4644ZsI4KUxNi7oUPMTFJObdmXiVgCLcB/s1600/Berende16-f2.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="316" src="https://3.bp.blogspot.com/-B3zrWIjfx-8/VxQ92ovg0wI/AAAAAAAAAqU/Wu-R4644ZsI4KUxNi7oUPMTFJObdmXiVgCLcB/s320/Berende16-f2.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Change in mean SF-36 physical component summary scores before and after treatment period. Figure 2 from Berende <i>et al.</i>, 2016.</td></tr>
</tbody></table>
<br />
Why wasn't a true placebo group? The authors worried about withholding antibiotics from subjects who might have an infection that should be treated. 11% of the subjects hadn't been treated with antibiotics for their symptoms prior to their acceptance into the study. This wasn't an issue with the earlier U.S. trials since previous treatment of Lyme disease with antibiotics was a requirement for acceptance into those studies, which included true placebo groups.<br />
<br />
One baffling aspect of the study was the inclusion of subjects who might not have had Lyme disease prior to the appearance of their chronic symptoms. Only a third of the subjects had objective clinical features of Lyme disease (erythema migrans, meningoradiculitis, or acrodermatitis chronica atrophicans) immediately preceding their chronic symptoms, and a little more than a half recalled a tick bite. Contrast this with the earlier U.S. studies, which only accepted patients with chronic symptoms
that followed antibiotic treatment of a well-documented case of Lyme disease.<br />
<br />
The remaining subjects did not have any objective features of Lyme disease before their chronic symptoms appeared. The only evidence of a previous episode of Lyme disease was positive antibody testing by Western blot. However, the antibodies may have been elicited by a <i>Borrelia</i> infection in the distant past. Their past episode of Lyme disease may not be related to their chronic symptoms, which aren't specific for Lyme disease. Another problem with relying solely on Western blots to diagnose Lyme disease is that false positives occur. Without additional evidence, it is hard to be sure that their chronic symptoms were related to Lyme disease.<br />
<br />
Nevertheless, the editorial accompanying the paper expressed support for the relaxed inclusion criteria used to select the subjects for the study:<br />
<blockquote>
Critics may rightly say that this trial does not
truly capture with certainty the consequences of
bona fide Lyme disease. However, studies with
more stringent inclusion criteria have already been
conducted, and the approach used by Berende
and colleagues probably reflects the common
practice in ambulatory care settings, in which
patient presentations of fatigue or nonspecific
pain prompt serologic checks for Lyme disease,
despite evidence suggesting that these tests will
not identify a probable cause or result in a treatment
benefit.</blockquote>
The study population may reflect what's encountered by clinicians in the real world, but for a clinical trial it doesn't seem right to lump those whose chronic problems followed a real episode of Lyme disease with those whose issues had nothing to do with Lyme. Any benefit of the antibiotics experienced by those who had genuine Lyme disease (assuming that there was any benefit) may have been obscured by the lack of benefit in those whose chronic symptoms aren't related to Lyme disease.<br />
<br />
Berende and colleagues also defended the length of treatment, which is considered to be on the short end by those who support lengthy courses of antibiotic therapy:<br />
<blockquote>
...it may be argued that 14 weeks of
treatment is insufficient to show a beneficial
treatment effect. However, whereas prolonged
antimicrobial treatment is not uncommon for
various infectious diseases, the purpose of
prolonged therapy for such diseases is for the
prevention of microbiologic relapse rather than
for a delayed onset of clinical alleviation of signs
or symptoms. We are not aware of any infectious
disease in which the initial effect on signs,
symptoms, and laboratory findings is delayed
beyond the first 3 months of effective therapy.
</blockquote>
But the graph above clearly shows that the subjects felt better following the treatment period. Unfortunately, as I mentioned earlier, the improvement in the quality of life can't be attributed to the antibiotics because there was no true placebo group.<br />
<br />
With these issues, I'm not sure how this study got published in NEJM. Regardless of my opinion, it will undoubtedly be cited as further proof that long-term antibiotics don't alleviate long-term symptoms that stem from Lyme disease.<br />
<br />
<i>Edit: Corrected quotes in first paragraph.</i> <br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+New+England+Journal+of+Medicine&rft_id=info%3Apmid%2F27028911&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Randomized+Trial+of+Longer-Term+Therapy+for+Symptoms+Attributed+to+Lyme+Disease.&rft.issn=0028-4793&rft.date=2016&rft.volume=374&rft.issue=13&rft.spage=1209&rft.epage=20&rft.artnum=&rft.au=Berende+A&rft.au=ter+Hofstede+HJ&rft.au=Vos+FJ&rft.au=van+Middendorp+H&rft.au=Vogelaar+ML&rft.au=Tromp+M&rft.au=van+den+Hoogen+FH&rft.au=Donders+AR&rft.au=Evers+AW&rft.au=Kullberg+BJ&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology%2C+Clinical+Research">Berende A, ter Hofstede HJ, Vos FJ, van Middendorp H, Vogelaar ML, Tromp M, van den Hoogen FH, Donders AR, Evers AW, & Kullberg BJ (2016). Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. <span style="font-style: italic;">The New England Journal of Medicine, 374</span> (13), 1209-20 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/27028911" rev="review">27028911</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+New+England+Journal+of+Medicine&rft_id=info%3Apmid%2F27028918&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Time+for+a+Different+Approach+to+Lyme+Disease+and+Long-Term+Symptoms.&rft.issn=0028-4793&rft.date=2016&rft.volume=374&rft.issue=13&rft.spage=1277&rft.epage=8&rft.artnum=&rft.au=Melia+MT&rft.au=Auwaerter+PG&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology%2C+Clinical+Research">Melia MT, & Auwaerter PG (2016). Time for a Different Approach to Lyme Disease and Long-Term Symptoms. <span style="font-style: italic;">The New England Journal of Medicine, 374</span> (13), 1277-8 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/27028918" rev="review">27028918</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-54036061851554338692016-03-21T18:20:00.001-07:002016-03-21T18:20:33.394-07:00The genomes of 20 species of LeptospiraA <a href="http://dx.doi.org/10.1371/journal.pntd.0004403" target="_blank">massive study</a> describing the genomes of 20 species of <i>Leptospira</i> was published a few weeks ago in <cite>PLOS Neglected Tropical Diseases</cite>. The deluge of sequence information will be valuable to those in the leptospirosis field. Scientists will be able to examine differences in genetic content between various categories of <i>Leptospira</i> species to generate hypotheses for experimental testing. For example, genes present in species that cause infections but missing in species that don't may be important factors responsible for the pathogenesis of <i>Leptospira</i>. The genome information will also aid in vaccine and serodiagnostics development by allowing researchers to identify protein antigens that are conserved among <i>Leptospira</i> species circulating within a region of interest.<br />
<br />
The 20 <i>Leptospira</i> species are divided into 14 infectious and six noninfectous species. (Actually, there are now 22 species known but only 20 when this study was launched.) The infectious species are divided further into nine pathogenic and five "intermediate" species based on their genetic relatedness. <br />
<br />
The Venn diagram below shows the number of genes that are shared among and within the three categories of <i>Leptospira</i> and <i>Leptonema illini</i>, a closely-related spirochete. Looking at the relevant intersection (overlap) in the diagram, there are 255 genes that are carried by infectious <i>Leptospira</i> but not by saprophytic <i>Leptospira</i>. (The other two figures in the overlap are the number of shared genes tabulated using looser criteria. In these cases there are 302 genes found in all but one infectious <i>Leptospira</i> and 369 genes when those found in the majority of infectious species are counted.) Similarly, there are 109 genes unique to the pathogenic species (or 161 or 416, if you want to use less stringent criteria). The small circles at the periphery show the number of genes unique to each species. So for example, <i>L. interrogans</i>, the species favored for study in molecular biology labs, has 672 genes that are not found in any other <i>Leptospira</i> species.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://2.bp.blogspot.com/-KcoOIZrFqQk/VtztUEtOwrI/AAAAAAAAAqA/SoCWTuKtVPc/s1600/Fouts16-f2a%2528shrink%2529.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="300" src="https://2.bp.blogspot.com/-KcoOIZrFqQk/VtztUEtOwrI/AAAAAAAAAqA/SoCWTuKtVPc/s400/Fouts16-f2a%2528shrink%2529.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2A from Fouts <i>et al.</i>, 2016. <a href="http://dx.doi.org/10.1371/journal.pntd.0004403.g002" target="_blank">Source</a>.</td></tr>
</tbody></table>
<br />
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+Neglected+Tropical+Diseases&rft_id=info%3Apmid%2F26890609&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=What+makes+a+bacterial+species+pathogenic%3F%3A+Comparative+genomic+analysis+of+the+genus+Leptospira.&rft.issn=1935-2727&rft.date=2016&rft.volume=10&rft.issue=2&rft.spage=&rft.epage=&rft.artnum=&rft.au=Fouts+DE&rft.au=Matthias+MA&rft.au=Adhikarla+H&rft.au=Adler+B&rft.au=Amorim-Santos+L&rft.au=Berg+DE&rft.au=Bulach+D&rft.au=Buschiazzo+A&rft.au=Chang+YF&rft.au=Galloway+RL&rft.au=Haake+DA&rft.au=Haft+DH&rft.au=Hartskeerl+R&rft.au=Ko+AI&rft.au=Levett+PN&rft.au=Matsunaga+J&rft.au=Mechaly+AE&rft.au=Monk+JM&rft.au=Nascimento+AL&rft.au=Nelson+KE&rft.au=Palsson+B&rft.au=Peacock+SJ&rft.au=Picardeau+M&rft.au=Ricaldi+JN&rft.au=Thaipandungpanit+J&rft.au=Wunder+EA+Jr&rft.au=Yang+XF&rft.au=Zhang+JJ&rft.au=Vinetz+JM&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA Jr, Yang XF, Zhang JJ, & Vinetz JM (2016). What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira. <span style="font-style: italic;">PLoS Neglected Tropical Diseases, 10</span> (2) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/26890609" rev="review">26890609</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-55964837467276525002016-02-11T11:47:00.000-08:002016-02-11T14:42:19.903-08:00How a new species of Lyme disease bacteria was discoveredA new agent of the tick-borne illness known as Lyme disease has emerged in the upper Midwest. The bacterium is genetically related to <i>Borrelia burgdorferi</i>, until now believed to be the only cause of Lyme disease in the United States. The name proposed for the bacterium is <i>Borrelia mayonii</i> because the work was conducted at the Mayo Clinic. <i>B. mayonii</i> has not been detected in patients outside of the Midwest (so far). The findings are described in <a href="http://dx.doi.org/10.1016/S1473-3099(15)00464-8" target="_blank"><cite>The Lancet Infectious Diseases</cite></a>.<br />
<br />
The new species was discovered at the Mayo Clinic during routine testing of specimens (blood, cerebral spinal fluid, and joint fluid) received from all regions of the U.S. Over 100,000 specimens collected from 2003 through 2014 were tested for Lyme disease bacteria by <a href="https://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction" target="_blank">real-time PCR</a> . The PCR probes were designed to detect the <i>oppA1</i> gene from <i>Borrelia</i> species belonging to the Lyme disease group, known in the scientific literature as "<i>B. burgdorferi</i> sensu lato." The Lyme disease group comprises 18 species that fall into the same genetic cluster within the genus <i>Borrelia</i>. They include species known or suspected to cause Lyme disease (<i>B. burgdorferi</i>, <i>B. garinii</i>, <i>B. afzelii</i>,<i> B. spielmanii</i>, <i>B. valaisiana</i>, <i>B bissettii</i>, <i>B. bavariensis</i>, and <i>B. lusitaniae</i>) and another ten species that do not cause illness. The PCR probes do not react with DNA from species belonging to the other cluster of <i>Borrelia</i>, the relapsing fever group.<br />
<br />
The key to the discovery of the new species was the melting temperature analysis routinely programmed onto the end of real-time PCR runs. The <i>oppA1</i> PCR products amplified from <i>B. burgdorferi</i> strains have melting temperatures of 63.6 through 64.9°C. For other Lyme disease species, the melting temperature ranges from 52.3°C (<i>B. valaisiana</i>) to 59.2°C (<i>B. californiensis</i>). Therefore, the melting temperature of the <i>oppA1</i> PCR product was used to distinguish <i>B. burgdorferi</i> from other Lyme disease <i>Borrelia.</i><br />
<br />
Over 9,000 specimens were collected from the states of Minnesota, Wisconsin, and North Dakota from January 2012 through September 2014. 102 were PCR positive, and most of the PCR products had the melting temperature profile of <i>B. burgdorferi</i>. However, six had melting temperatures ranging from 60.4°C to 61.2°C, too low to be <i>B. burgdorferi</i> but too high to be any other member of the Lyme disease group. The novel spirochetes were cultured from the blood of two of the patients. The DNA sequence of several "housekeeping" genes of the new isolates differed enough from those of other <i>Borrelia</i> species to signify that a new <i>Borrelia</i> species has been found. The investigators named the new spirochete <i>Borrelia mayonii</i>. No specimen collected from other regions of the U.S. exhibited the
atypical melting temperatures, and neither did any collected
earlier than 2012 from the Midwest. These findings led the authors to conclude that <i>B. mayonii</i> has recently emerged in the upper Midwest and that the six patients are the first known cases of Lyme disease to be caused by the new species.<br />
<br />
The investigators also collected <i>Ixodes scapularis</i> ticks in Wisconsin. PCR and melting temperature analysis showed that 19 of 658 ticks (2.9%) were positive for <i>B. mayonii</i>, 195 (29.6%) positive for <i>B. burgdorferi</i>, and two positive for both.<br />
<br />
One striking feature of <i>B. mayonii</i> infections is the large number of spirochetes circulating within the patients. The densities ranged from 420,000 to 6,400,000 bacterial cells per milliliter, at least a hundred times higher than observed in the blood of patients with <i>B. burgdorferi</i> infections. The numbers were high enough that spirochetes could be seen in blood collected from one of the patients.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://4.bp.blogspot.com/-N133Zv_Fk1g/VrtyhR-JpyI/AAAAAAAAAps/81OYkVFYM8o/s1600/Pritt-f1b.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://4.bp.blogspot.com/-N133Zv_Fk1g/VrtyhR-JpyI/AAAAAAAAAps/81OYkVFYM8o/s1600/Pritt-f1b.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fig. 1b from Pritt et al., 2016</td></tr>
</tbody></table>
<br />
The six patients had many of the typical Lyme disease symptoms: headache, neck pain, muscle aches, joint pain, and fatigue. Although mild fever is also common in Lyme disease, two of the six patients had severe fevers with temperature readings approaching 40°C (104°F). Four had nausea or were vomiting, which are also uncommon Lyme disease symptoms. Two patients were hospitalized because of the severity of their illness. Lyme disease may be missed in those infected with <i>B. mayonii</i> because of the unusual symptoms.<br />
<br />
The standard <a href="http://www.cdc.gov/lyme/healthcare/clinician_twotier.html" target="_blank">two-tier antibody test</a>, which uses <i>B. burgdorferi</i> antigens to detect reactive antibody, may help with the diagnosis. Blood specimens from five of the six patients were tested. Four patients either tested positive or, if negative initially, tested positive with blood drawn weeks later. The one patient who tested negative had blood drawn only on the first day of illness, so it's likely that the antibody response hadn't kicked in fully. The test appears to help with the diagnosis of Lyme disease caused by <i>B. mayonii</i>, but the number of patients tested was too small to draw firm conclusions.<br />
<br />
The authors conclude:<br />
<br />
<blockquote>
In view of
the differing clinical manifestations for patients infected
with the novel <i>B burgdorferi</i> sensu lato genospecies, it is
likely that Lyme borreliosis is not being considered—and
therefore not diagnosed—in some patients with this
infection. The clinical range of illness must be better
defined in additional patients to ensure that physicians
can recognise the infection and distinguish it from other
tick-borne infections. Many tick-borne pathogens have
global distribution, therefore studies are needed to
establish the geographic distribution of human beings
and ticks infected with the novel <i>B. burgdorferi</i> sensu lato
genopecies. Finally, clinicians should be aware of the
potential role of <i>oppA1</i> PCR for diagnosing infection with
this novel pathogen.</blockquote>
<br />
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Lancet.+Infectious+diseases.&rft_id=info%3Apmid%2F26856777&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Identification+of+a+novel+pathogenic+Borrelia+species+causing+Lyme+borreliosis+with+unusually+high+spirochaetaemia%3A+a+descriptive+study.&rft.issn=1473-3099&rft.date=2016&rft.volume=&rft.issue=&rft.spage=&rft.epage=&rft.artnum=&rft.au=Pritt+BS&rft.au=Mead+PS&rft.au=Johnson+DK&rft.au=Neitzel+DF&rft.au=Respicio-Kingry+LB&rft.au=Davis+JP&rft.au=Schiffman+E&rft.au=Sloan+LM&rft.au=Schriefer+ME&rft.au=Replogle+AJ&rft.au=Paskewitz+SM&rft.au=Ray+JA&rft.au=Bjork+J&rft.au=Steward+CR&rft.au=Deedon+A&rft.au=Lee+X&rft.au=Kingry+LC&rft.au=Miller+TK&rft.au=Feist+MA&rft.au=Theel+ES&rft.au=Patel+R&rft.au=Irish+CL&rft.au=Petersen+JM&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology%2C+Taxonomy">Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL, & Petersen JM (2016). Identification of a novel pathogenic <i>Borrelia</i> species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. <span style="font-style: italic;">The Lancet. Infectious diseases.</span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/26856777" rev="review">26856777</a></span>
Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-26399916606092312352015-11-11T23:45:00.000-08:002015-11-11T23:45:09.210-08:00Lighting up Leptospira interrogans to test antibiotic treatment of chronically-infected miceA group at the Pasteur Institute succeeded in generating a bioluminescent strain of <i>Leptospira interrogans</i>. Their study was <a href="http://dx.doi.org/10.1371/journal.pntd.0003359" target="_blank">published</a> last year in <cite>PLOS Neglected Tropical Diseases</cite>.<br />
<br />
The benefit of having a bioluminescent strain is that infections of small laboratory rodents can be monitored without sacrificing the animals. Instead, the animals are placed in a special whole-body imager that detects light emitted from the bodies. The location of the infection in the animals can be determined from the images, and the light intensity measured by the imager gives an idea of the bacterial load in infected tissues. After imaging, the animal can be returned to its cage, and additional images of the same animal can be taken later as the infection progresses.<br />
<br />
Another advantage of using bioluminescent bacteria is that the luciferase reaction requires ATP, meaning that the bacteria must be metabolically active to light up. Dead bacteria containing luciferase will not generate light, unlike green fluorescent protein (GFP), another reporter used to image bacteria (see <a href="http://spirochetesunwound.blogspot.com/2012/11/inflammatory-spirochete-debris-left.html" target="_blank">this</a> post that describes an experiment with <i>Borrelia burgdorferi</i> expressing GFP).<br />
<br />
To generate bioluminescent <i>L. interrogans</i>, the researchers hooked the firefly <a href="https://en.wikipedia.org/wiki/Luciferase" target="_blank">luciferase</a> gene up to a strong <i>L. interrogans</i> promoter and inserted the construct into a transposon carried on a suicide plasmid. The plasmid was then introduced into <i>L. interrogans</i> by conjugation (see <a href="http://spirochetesunwound.blogspot.com/2009/12/leptospira-and-e-coli-caught-in-act.html" target="_blank">this</a> blog post for details of the process). Cultures of the engineered spirochetes lit up when <a href="https://en.wikipedia.org/wiki/Luciferin" target="_blank">luciferin</a>, the luciferase substrate, was added. The amount of light emitted depended on the culture density: more light was detected at higher culture densities.<br />
<br />
Mice are ideal models to study persistent infection of the kidney since many rodents are chronic carriers of <i>Leptospira</i> out in nature. These rodents don't get sick from the infection, but they contaminate the environment with the spirochete every time they urinate.<br />
<br />
To see how chronic infection is established, the investigators injected a sublethal dose of 10<sup>7</sup> bioluminescent <i>L. interrogans</i> cells into the abdominal cavity of C57BL6/J mice and took sequential images of the animals over the following months. Albino mice were used because dark fur blocks the signal emitted by the bioluminescent bacteria. (They later showed that standard C57BL6/J mice with black fur could be used as long as they shaved the fur off before placing them in the imager.) The mice were injected with luciferin 10 minutes prior to imaging. <br />
<br />
There turned out to be two phases of infection (see the "MFlum1" plot in the graph below). In the acute phase, the bioluminescent signal rose to a peak by day 4 and quickly declined to background levels by day 7. The signal then started increasing again slowly and plateaued after a month.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-5jNUfuKbMuE/Vgh8ZHnoSXI/AAAAAAAAAoE/wZbS8znegBU/s1600/Ratet14-f2a%2528small%2529.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="212" src="http://3.bp.blogspot.com/-5jNUfuKbMuE/Vgh8ZHnoSXI/AAAAAAAAAoE/wZbS8znegBU/s400/Ratet14-f2a%2528small%2529.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2A from Ratet <i>et al.</i>, 2014. Images of a single mouse taken sequentially are shown below the graph. Click for larger image. <a href="http://dx.doi.org/10.1371/journal.pntd.0003359.g002" target="_blank">Source</a>.</td></tr>
</tbody></table>
Images of a single mouse taken at different times after inoculation are shown below the graph. Thirty minutes after inoculation, signal was detected in the abdominal cavity. By day 3, the signal consumed the entire mouse. At this point, the bacteria were probably circulating in the bloodstream. By day 6, the signal was almost completely gone. After day 6, the signal appeared again, but it was confined to the kidneys. The intensity of the signal in the kidneys increased with time. They did not detect signal anywhere else in the animals during the second phase. They even sacrificed some of the infected mice 2 months into the infection to check the organs directly, but they failed to detect <i>Leptospira</i> by bioluminescence and qPCR in the brain, lungs, spleen, liver, or blood. Not surprisingly, bioluminescence was detected in urine, confirming that the mice were shedding live <i>L. interrogans</i>.<br />
<br />
Next, the investigators tested the effectiveness of antibiotics in treating mice infected with the bioluminescent <i>L. interrogans</i>. Several antibiotics are used to treat acute leptospirosis in humans, including penicillin and azithromycin. It is generally believed that antibiotics are more effective if provided early in acute illness. Therefore, the investigators tested whether the timing of antibiotic treatment was important for effectiveness.<br />
<br />
As expected, penicillin treatment was most effective when treatment was started at the beginning of the acute phase. In mice treated with daily injections of penicillin for 5 days starting a day after infection, no bioluminescence was detected in the kidneys, and urine was free of <i>L. interrogans</i> as measured by qPCR. However, if treatment was delayed until three days after inoculation, during the peak of acute infection, a low level of <i>L. interrogans</i> was detected in urine by qPCR even though no bioluminescence was detected in the kidneys. It is likely <i>L. interrogans</i> was present in the kidneys but at levels too low to be detected by the imager. The bioluminescence approach clearly does not have the sensitivity of qPCR. Additional experiments revealed that the limit of detection was 100 bioluminescent <i>L. interrogans</i> cells in 100 μl of buffer.<br />
<br />
Penicillin was even less effective when administered after the spirochetes settled in the kidneys. When penicillin treatment was initiated at peak bacterial load in the kidneys, day 25 of infection, the signal diminished by over 90% but then bounced back to the level observed before treatment began (see figure below). Ciprofloxacin also failed to eradicate the bacteria.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-bH6v616w_zM/VkP86skzwwI/AAAAAAAAAos/uHsP61MyawU/s1600/Ratet14-f5a.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-bH6v616w_zM/VkP86skzwwI/AAAAAAAAAos/uHsP61MyawU/s1600/Ratet14-f5a.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5A from Ratet <i>et al.</i>, 2014. Antibiotics were administered for 5 days started on day 25 of infection. Cipr, ciprofloxacin; Pen, penicillin. <a href="http://dx.doi.org/10.1371/journal.pntd.0003359.g005" target="_blank">Source</a>.</td></tr>
</tbody></table>
<br />
On the other hand, azithromycin managed to knock the signal in the kidneys down to background levels (see graph below). However, the signal came back within a week, although not to the high levels seen in untreated mice. A second course of antibiotics starting on day 112 knocked the signal back down to near background levels, but again, spirochete numbers rebounded, although not to the levels seen before retreatment.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://4.bp.blogspot.com/-nofNtLQF8i4/VkP9T43vpjI/AAAAAAAAAo0/iNwKHJGK7fs/s1600/Ratet14-f5b.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://4.bp.blogspot.com/-nofNtLQF8i4/VkP9T43vpjI/AAAAAAAAAo0/iNwKHJGK7fs/s1600/Ratet14-f5b.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5B from Ratet <i>et al.</i>, 2014. Azithromycin was administered for 5 days starting on day 25 and day 112 of infection. <a href="http://dx.doi.org/10.1371/journal.pntd.0003359.g005" target="_blank">Source</a>.</td></tr>
</tbody></table>
Why are antibiotics ineffective in eradicating <i>L. interrogans</i> during the chronic phase? Like other bacteria, <i>L. interrogans</i> can form biofilms <i>in vitro</i>. Scientists who work with <i>Leptospira</i> believe that they also assemble into biofilms within the kidney tubules during chronic infection. Biofilms are hard to eliminate in part because they harbor persister cells that tolerate antibiotics. (See <a href="http://spirochetesunwound.blogspot.com/2012/02/magic-of-antibiotic-tolerance.html" target="_blank">this</a> post for some background on persister cells.)<br />
<br />
I should caution readers from concluding that tolerance accounts for the poor effectiveness of antibiotics in treating human cases of <i>acute</i> leptospirosis. As the authors point out, leptospirosis patients die because the infection severely injure vital organs. By the time lethal damage occurs, it does not matter whether antibiotics kill all of the spirochetes.<br />
<br />
So does the mouse model have any relevance to human leptospirosis? The authors argue that asymptomatic carriage of <i>Leptospira</i> has been overlooked. A <a href="http://dx.doi.org/10.1371/journal.pone.0076549" target="_blank">2013 study</a> from the Netherlands revealed that 21% of patients who contracted leptospirosis continued to suffer from headaches, muscle aches, and extreme fatigue two years later. This may reflect unrepaired tissue damage inflicted during acute infection, but no one checked for the presence of <i>Leptospira</i> in these patients. Another study from Peru (see <a href="http://spirochetesunwound.blogspot.com/2010/04/healthy-human-carriers-of-spirochete.html" target="_blank">this</a> post) describes asymptomatic individuals who may have persistent <i>Leptospira</i> infection. Kidney function was not checked in the Peruvians, but there is reason to believe that chronic infection affects the kidneys despite the lack of symptoms. Mice chronically infected with <i>L. interrogans</i> are not visibly sick, but they end up with scarred kidneys (<a href="https://en.wikipedia.org/wiki/Fibrosis" target="_blank">fibrosis</a>), as explained in <a href="http://dx.doi.org/10.1371/journal.pntd.0002664" target="_blank">this</a> study.<br />
<br />
If persistent asymptomatic infections really do occur in humans, it may be sensible to treat with antibiotics. The chronically-infected mouse will serve as a nice model for testing antibacterial regimens that target <i>Leptospira</i> living in the kidneys.<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+Neglected+Tropical+Diseases&rft_id=info%3Apmid%2F25474719&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Live+imaging+of+bioluminescent+Leptospira+interrogans+in+mice+reveals+renal+colonization+as+a+stealth+escape+from+the+blood+defenses+and+antibiotics.&rft.issn=1935-2727&rft.date=2014&rft.volume=8&rft.issue=12&rft.spage=&rft.epage=&rft.artnum=&rft.au=Ratet+G&rft.au=Veyrier+FJ&rft.au=Fanton+d%27Andon+M&rft.au=Kammerscheit+X&rft.au=Nicola+MA&rft.au=Picardeau+M&rft.au=Boneca+IG&rft.au=Werts+C&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Ratet G, Veyrier FJ, Fanton d'Andon M, Kammerscheit X, Nicola MA, Picardeau M, Boneca IG, & Werts C (2014). Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics. <span style="font-style: italic;">PLoS Neglected Tropical Diseases, 8</span> (12) PMID: <a rev="review" href="http://www.ncbi.nlm.nih.gov/pubmed/25474719">25474719</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PloS+One&rft_id=info%3Apmid%2F24098528&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Towards+the+burden+of+human+leptospirosis%3A+duration+of+acute+illness+and+occurrence+of+post-leptospirosis+symptoms+of+patients+in+the+Netherlands.&rft.issn=&rft.date=2013&rft.volume=8&rft.issue=10&rft.spage=&rft.epage=&rft.artnum=&rft.au=Goris+MG&rft.au=Kikken+V&rft.au=Straetemans+M&rft.au=Alba+S&rft.au=Goeijenbier+M&rft.au=van+Gorp+EC&rft.au=Boer+KR&rft.au=Wagenaar+JF&rft.au=Hartskeerl+RA&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Goris MG, Kikken V, Straetemans M, Alba S, Goeijenbier M, van Gorp EC, Boer KR, Wagenaar JF, & Hartskeerl RA (2013). Towards the burden of human leptospirosis: duration of acute illness and occurrence of post-leptospirosis symptoms of patients in the Netherlands. <span style="font-style: italic;">PloS One, 8</span> (10) PMID: <a rev="review" href="http://www.ncbi.nlm.nih.gov/pubmed/24098528">24098528</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+Neglected+Tropical+Diseases&rft_id=info%3Apmid%2F24498450&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Leptospira+Interrogans+induces+fibrosis+in+the+mouse+kidney+through+Inos-dependent%2C+TLR-+and+NLR-independent+signaling+pathways.&rft.issn=1935-2727&rft.date=2014&rft.volume=8&rft.issue=1&rft.spage=&rft.epage=&rft.artnum=&rft.au=Fanton+d%27Andon+M&rft.au=Quellard+N&rft.au=Fernandez+B&rft.au=Ratet+G&rft.au=Lacroix-Lamand%C3%A9+S&rft.au=Vandewalle+A&rft.au=Boneca+IG&rft.au=Goujon+JM&rft.au=Werts+C&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Fanton d'Andon M, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, Boneca IG, Goujon JM, & Werts C (2014). Leptospira Interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR- and NLR-independent signaling pathways. <span style="font-style: italic;">PLoS Neglected Tropical Diseases, 8</span> (1) PMID: <a rev="review" href="http://www.ncbi.nlm.nih.gov/pubmed/24498450">24498450</a></span><br />
<br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2011/10/life-after-leptospirosis-pilot-study.html" target="_blank">Life after leptospirosis, a pilot study</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2010/04/healthy-human-carriers-of-spirochete.html" target="_blank">Healthy human carriers of the spirochete <i>Leptospira</i> in the Peruvian Amazon</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2012/02/magic-of-antibiotic-tolerance.html" target="_blank">The magic of antibiotic tolerance</a></li>
</ul>
<br />
<br />
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-26421628922073889592015-09-06T18:28:00.000-07:002015-09-06T21:05:59.127-07:00Do Lyme disease spirochetes produce a toxin?According to the current view of Lyme disease pathogenesis, tissue damage is caused by the inflammatory response to the spirochetes. <i>Borrelia</i> species do not produce toxins that injure the host directly. A <a href="http://dx.doi.org/10.1186/s12866-015-0464-y" target="_blank">new study</a> published in <cite>BMC Microbiology</cite> may force us to modify our view.<br />
<br />
The study shows that some <i>Borrelia</i> strains carry a set of genes with the potential to generate a peptide resembling streptolysin S (SLS), a potent toxin produced by the pathogen <i>Streptococcus pyogenes</i>. The enzymes that produce SLS in <i>S. pyogenes</i> are expressed from a cluster of genes surrounding <i>sagA</i>, a tiny gene encoding the SLS precursor. The peptide produced from <i>sagA</i> is nontoxic; it has to undergo several alterations to its structure to become toxic. A critical modification is carried out by the SagBCD protein complex, which converts the side chains of cysteine, serine, and threonine into ring structures.<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-4sSyeC_T6I8/VelF-LM_O5I/AAAAAAAAAnI/BuHmPp7THmE/s1600/Molloy11-f2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="400" src="http://3.bp.blogspot.com/-4sSyeC_T6I8/VelF-LM_O5I/AAAAAAAAAnI/BuHmPp7THmE/s400/Molloy11-f2.jpg" width="222" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2 from Molloy <i>et al.</i>, 2011</td></tr>
</tbody></table>
<br />
Other genes surrounding <i>sagA</i> encode a peptidase that is thought to trim the leader peptide from the amino terminus of the SLS precursor and an <a href="https://en.wikipedia.org/wiki/ATP-binding_cassette_transporter" target="_blank">ABC transporter</a> that may be responsible for expelling SLS from the cytoplasm.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-A577u9HF5vo/VelG2V8sXrI/AAAAAAAAAnQ/IAmKeyUJI6c/s1600/Molloy11-f1a.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="280" src="http://3.bp.blogspot.com/-A577u9HF5vo/VelG2V8sXrI/AAAAAAAAAnQ/IAmKeyUJI6c/s400/Molloy11-f1a.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1A from Molloy <i>et al.</i>, 2011</td></tr>
</tbody></table>
<br />
SLS targets neutrophils and possibly other immune cells during <i>S. pyogenes</i> infection. SLS-like toxins are also produced by other Gram-positive pathogens, including <i>Staphylococcus aureus</i>, <i>Listeria monocytogenes</i> and <i>Clostridium botulinum</i>.<br />
<br />
The investigators mined the genomes of other bacteria in search for genes encoding the machinery that generates SLS-like toxins. They found SLS-like gene clusters in various <i>Firmicutes</i> and <i>Actinobacteria</i>, both Gram-positive groups of bacteria.<br />
<br />
The researchers also found the gene cluster in the genomes of <i>Borrelia afzelii</i> strain PKo, <i>Borrelia valaisiana</i> strain VS116, and <i>Borrelia spielmanii</i> strain A14S. <i>B. afzelii</i> is a major cause of Lyme disease in Europe and Asia. <i>B. valaisiana</i> and <i>B. spielmanii</i> are responsible for occasional cases of Lyme disease.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-66XEbX25RsM/VelIyuiXXcI/AAAAAAAAAnc/Oxakffcvpkw/s1600/Molloy15-f4ab.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="186" src="http://2.bp.blogspot.com/-66XEbX25RsM/VelIyuiXXcI/AAAAAAAAAnc/Oxakffcvpkw/s400/Molloy15-f4ab.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 from Molloy <i>et al.</i>, 2015. Top: organization of SLS-like gene cluster in <i>S. pyogenes</i> and three <i>Borrelia</i> strains. Bottom: sequence of the SLS precursor (SagA) and the borrelial SLS-like precursors.</td></tr>
</tbody></table>
<br />
They also used PCR to screen the DNA of 140 patient and tick isolates of Lyme <i>Borrelia</i> for the genes encoding the SLS-like biosynthetic machinery. Most of the isolates were obtained from Europe and the U.S., with a few coming from Asia. Design of the PCR primers was based on the sequence of the <i>B. valaisiana</i> <i>bvalB</i>, <i>bvalC</i>, and <i>bvalD</i> genes, which encode homologs of the <i>S. pyogenes</i> <i>sagB</i>, <i>sagC</i>, and <i>sagD</i> gene products. Most of the <i>B. garinii</i>, <i>B. afzelii</i>, <i>B. valaisiana</i>, <i>B. spielmanii</i>, and <i>B. lusitaniae</i> isolates that were examined tested positive. On the other hand, none of the 22 isolates of <i>B. burgdorferi</i> or 13 isolates of <i>B. bavariensis</i> were PCR positive. These results indicate that SLS-like sequences are widespread among Lyme disease spirochetes (though not in <i>B. burgdorferi</i>).<br />
<br />
The next step was to show that the SLS-like borrelial gene actually encoded a peptide that damages mammalian cells. A simple assay based on the ability of many toxins to rupture (hemolyze) red blood cell <i>in vitro</i> is available. Hemolysis is measured easily by mixing the toxin with sheep red blood cells. Hemoglobin released from the ruptured cells is quantified with a spectrophotometer.<br />
<br />
They decided to test the SLS-like peptide encoded by <i>B. valaisiana</i>, BvalA, for hemolytic activity. The researchers succeeded in expressing and purifying a recombinant form of BvalA. Not surprisingly, BvalA was not hemolytic because its amino acid side chains had to be converted into ring structures necessary for the peptide to injure red blood cells. They wanted to mix BvalA with the BvalBCD protein complex so that the peptide would be modified, but they could not generate the protein complex. Instead, they used the SagBCD complex from <i>S. pyogenes</i> to modify the BvalA peptide. When they did this, they finally observed hemolytic activity.<br />
<br />
Red blood cells are unlikely to be a major target of borrelial SLS-like peptides during infection. So what is the real target? More studies are needed to answer this question, but we should consider the possibility that the toxin has nothing to do with Lyme disease. Instead, it may help the spirochete to survive during its residence within the tick vector. A number of nonpathogenic bacteria carry gene clusters distantly related to the ones that produce SLS. Several peptide toxins produced by these bacteria are known to kill competing microbes. Like us humans, ticks have a microbiome inhabiting their gut. Some Lyme spirochetes may need to secrete the toxin to ward off their microbial neighbors.<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=BMC+Microbiology&rft_id=info%3Apmid%2F26204951&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Identification+of+the+minimal+cytolytic+unit+for+streptolysin+S+and+an+expansion+of+the+toxin+family.&rft.issn=&rft.date=2015&rft.volume=15&rft.issue=&rft.spage=141&rft.epage=&rft.artnum=&rft.au=Molloy+EM&rft.au=Casjens+SR&rft.au=Cox+CL&rft.au=Maxson+T&rft.au=Ethridge+NA&rft.au=Margos+G&rft.au=Fingerle+V&rft.au=Mitchell+DA&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Molloy EM, Casjens SR, Cox CL, Maxson T, Ethridge NA, Margos G, Fingerle V, & Mitchell DA (2015). Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family. <span style="font-style: italic;">BMC Microbiology, 15</span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/26204951" rev="review">26204951</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Nature+Reviews+Microbiology&rft_id=info%3Apmid%2F21822292&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Streptolysin+S-like+virulence+factors%3A+the+continuing+sagA.&rft.issn=1740-1526&rft.date=2011&rft.volume=9&rft.issue=9&rft.spage=670&rft.epage=81&rft.artnum=&rft.au=Molloy+EM&rft.au=Cotter+PD&rft.au=Hill+C&rft.au=Mitchell+DA&rft.au=Ross+RP&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Molloy EM, Cotter PD, Hill C, Mitchell DA, & Ross RP (2011). Streptolysin S-like virulence factors: the continuing sagA. <span style="font-style: italic;">Nature Reviews Microbiology, 9</span> (9), 670-81 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21822292" rev="review">21822292</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com2tag:blogger.com,1999:blog-1574140332407591967.post-62290884710229396132015-08-17T21:18:00.000-07:002015-08-18T10:18:56.287-07:00A biosignature of early Lyme diseaseLaboratory testing for Lyme disease involves two-tier antibody testing with sera from patients suspected of having the disease. The first step is usually an <a href="https://en.wikipedia.org/wiki/ELISA" target="_blank">ELISA</a> with a cell lysate of <i>Borrelia burgdorferi</i> as antigen. If the ELISA results are positive or borderline, a Western blot is done to confirm that the patient has Lyme disease. Direct detection of <i>Borrelia burgdorferi</i> by culture would be the preferred laboratory test, but it takes too long for the spirochete to grow. Culture of patient specimens is done only for research studies.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://1.bp.blogspot.com/-fxp64Ps6FSA/VcgDRJU5IeI/AAAAAAAAAmk/8p0VN7nsU70/s1600/CDC-2TierTest.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="300" src="http://1.bp.blogspot.com/-fxp64Ps6FSA/VcgDRJU5IeI/AAAAAAAAAmk/8p0VN7nsU70/s400/CDC-2TierTest.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Source: <a href="http://www.cdc.gov/lyme/diagnosistesting/labtest/twostep/index.html" target="_blank">CDC</a></td></tr>
</tbody></table>
In general, the problem with antibody testing for infectious diseases is that it takes time for the immune system to generate antibody against the pathogen. Therefore, patients in the early stages of infection may test negative. False-negative tests may delay appropriate treatment until the illness worsens. For these reasons, scientists have been trying to come up with better laboratory tests for infections whose diagnosis relies on detecting antibody against the infectious agent.<br />
<br />
One new approach being developed for a few pathogens involves measuring the amounts of each of the thousands of small molecules found in the sera of infected patients. This is done by <a href="https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry" target="_blank">liquid chromatography/mass spectrometry</a> (LC-MS), which accurately and precisely measures the size of small molecules, even in complex substances like serum. The assumption is that the composition of small molecules (the so-called "<a href="https://en.wikipedia.org/wiki/Metabolome" target="_blank">metabolome</a>") starts to change in a predictable manner as soon as someone is infected. The metabolome changes because tissues react to the pathogen by generating inflammatory molecules that leak into the bloodstream. Another critical assumption is that the changes that occur in the metabolome are unique to each pathogen. Analysis of the patient's metabolome may therefore allow clinicians to quick diagnose any infectious disease whose metabolome has been characterized.<br />
<br />
A <a href="http://dx.doi.org/10.1093/cid/civ185" target="_blank">recent CDC study</a> revealed the metabolome of early Lyme disease. The investigators obtained sera from 89 patients who had early-stage Lyme disease. All had erythema migrans (EM), the rash characteristic of Lyme disease. Most were also culture or PCR positive for <i>Borrelia burgdorferi</i>. The patient sera were compared with sera from 50 healthy individuals by LC-MS.<br />
<br />
After two runs of LC-MS with each sample, the researchers identified a set of 95 small molecules whose levels consistently differed between patients with early Lyme disease and healthy individuals. Statistical modeling of the data allowed the investigators to refine the biosignature to a set of 44 molecules that identified Lyme disease in the 139 (89 + 50) subjects with the highest <a href="https://en.wikipedia.org/wiki/Sensitivity_and_specificity" target="_blank">sensitivity and specificity</a>.<br />
<br />
To better gauge the performance of the biosignature in identifying those with early Lyme disease, the investigators conducted LC-MS on sera from another group of 91 patients shown to have early Lyme disease by the same criteria as the first set of patients. Control sera came from 108 healthy individuals. Another set of control sera was obtained from 101 patients with other diseases that could be confused with Lyme disease clinically, serologically, or microbiologically: syphilis, severe periodontitis, infectious mononucleosis, and fibromyalgia. All patient and control sera were also tested by the standard two-tier antibody test.<br />
<br />
The sensitivity of LC-MS testing turned out to be much higher than that of two-tier testing: 88% vs. 44%. The specificity of LC-MS was 94% with healthy sera and 95% when sera from patients with other diseases were tested. These values were not significantly different from the specificities of 100% and 95% achieved with two-tier testing.<br />
<br />
These results show the promise of using the metabolic biosignature to help diagnose early Lyme disease. However, note that all patient sera used to uncover the biosignature and assess its performance came from individuals with EM. In practice, a clinical diagnosis involving the classic bulls-eye EM with a patient history suggestive of Lyme disease does not require confirmation by laboratory testing. Patients without EM are more likely to need laboratory testing. According to the CDC, <a href="http://wwwn.cdc.gov/nndss/conditions/lyme-disease/case-definition/2011/" target="_blank">20-40%</a> of Lyme disease patients do not have EM.<br />
<br />
To get an idea of how well the biosignature performs on patients without EM, the investigators obtained sera drawn from 22 cases with early Lyme disease who tested positive with the C6 ELISA, a newer antibody test. The antigen for the C6 ELISA is a highly-conserved peptide from the <i>B. burgdorferi</i> surface protein VlsE. Eight of the 22 patients did not have EM. The EM status was unknown in another eight patients. The remaining six patients had EM. Unfortunately, the results for each subgroup were not presented by the authors, so we don't have a firm answer about the performance of LC-MS testing on patients without EM. What we can say is that even though more than a third of the 22 patients did not have EM, the sensitivity of LC-MS testing remained high at 86%. In contrast, the sensitivity of two-tier testing with this group was only 41%, even though the investigators stacked the deck by using the C6 ELISA as the first tier with this group. Future testing of the biosignature should include a larger number of sera from EM-negative patients in the early stages of Lyme disease. <br />
<br />
As discussed in the paper, sera from patients with skin conditions that could be confused with EM (e.g., <a href="http://spirochetesunwound.blogspot.com/2009/07/stari-or-masters-disease-more-like-lyme.html" target="_blank">STARI</a>, cellulitis) should be examined in future studies to make sure that the early Lyme biosignature can be used to rule out those conditions. The authors recommend that sera from patients with neurologic, cardiac, and arthritic forms of Lyme disease also be examined to see if biosignatures specific for these more serious forms of Lyme disease could be identified.<br />
<b><br /></b>
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+Infectious+Diseases&rft_id=info%3Apmid%2F25761869&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Development+of+a+metabolic+biosignature+for+detection+of+early+Lyme+disease.&rft.issn=1058-4838&rft.date=2015&rft.volume=60&rft.issue=12&rft.spage=1767&rft.epage=1775&rft.artnum=&rft.au=Molins+CR&rft.au=Ashton+LV&rft.au=Wormser+GP&rft.au=Hess+AM&rft.au=Delorey+MJ&rft.au=Mahapatra+S&rft.au=Schriefer+ME&rft.au=Belisle+JT&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMedicine%2CMicrobiology">Molins CR, Ashton LV, Wormser GP, Hess AM, Delorey MJ, Mahapatra S, Schriefer ME, & Belisle JT (2015). Development of a metabolic biosignature for detection of early Lyme disease. <span style="font-style: italic;">Clinical Infectious Diseases, 60</span> (12), 1767-1775 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/25761869" rev="review">25761869</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-5021952541808978022014-12-30T14:50:00.000-08:002014-12-30T21:43:12.155-08:00Severe Lyme arthritis: Gagging on GAGs<a href="http://www.bioscience.utah.edu/faculty/molecular-biology-faculty/weisjanis/weisJanis.php#research" target="_blank">Janis Weis' group</a> has been mapping genetic variants that make laboratory mice prone to severe Lyme arthritis. One of these variants is described in a <a href="http://www.ncbi.nlm.nih.gov/pubmed/24334460" target="_blank">paper</a> that appeared in <cite>The Journal of Clinical Investigation</cite> earlier this year. The affected gene encodes the enzyme β-glucuronidase, which carries out a critical function in the <a href="http://en.wikipedia.org/wiki/Lysosome" target="_blank">lysosome</a>. β-glucuronidase cooperates with other degradative enzymes in the lysosome to break down <a href="http://www.ncbi.nlm.nih.gov/books/NBK26810/#A3537" target="_blank">glycosaminoglycans</a> (GAGs) into their individual sugar units, which are then removed from the lysosome and reused by the cells. GAGs are long chains of specific disaccharides located on the cell surface and within the extracellular matrix. GAGs are covalently (in <a href="http://www.ncbi.nlm.nih.gov/books/NBK1900/" target="_blank">proteoglycans</a>) or noncovalently attached to proteins. GAGs are always being degraded and resynthesized by cells. Blocking any of the enzymes involved in GAG breakdown causes accumulation of GAG fragments, which are potentially detrimental to health. In humans, certain mutations in the β-glucuronidase gene lead to a rare condition called <a href="http://ghr.nlm.nih.gov/condition/mucopolysaccharidosis-type-vii" target="_blank">Sly syndrome</a>.<br />
<br />
Large amounts of GAGs are found in the joints, where they serve an important mechanical function. GAGs carry a high density of negative charge due to the presence of acidic sugars such as glucuronic acid, the target of β-glucuronidase, and the sulfate groups attached to most types of GAGs. The negative charge attracts cations, which in turn
attract large numbers of water molecules. The water within GAGs acts as a cushion that allows
the joints to withstand large compressive forces.<br />
<br />
The key to the study was having strains of mice that differed in their susceptibility to Lyme arthritis. The C3H mouse strain develops severe joint inflammation during <i>B. burgdorferi</i> infection. On the other hand, the B6 strain develops mild joint inflammation when infected. Weis' group had earlier narrowed the locations of the genetic variations accounting for the different susceptibilities to several distinct segments within the mouse genome. They used the traditional techniques of mouse genetics, which involved numerous matings involving the C3H and B6 strains and their progeny (see <a href="http://www.ncbi.nlm.nih.gov/pubmed/24926442" target="_blank">this review</a> for details). The authors focused on one of the segments, and with help from mouse genome sequence data that became available, they found a nucleotide difference within the <i>Gusb</i> (β-glucuronidase) gene that changed a single amino acid in the enzyme.<br />
<br />
The investigators found that β-glucuronidase activity was mildly reduced in the infected C3H strain relative to the B6 strain. Staining tissue sections of infected mice with Alcian blue, a dye attracted to polyanions, revealed accumulation of GAGs in the joint tissues of infected C3H mice but not infected B6 mice, lending further support to the lesion in <i>Gusb</i> being responsible for severe inflammation. When a functional copy of the β-glucuronidase gene was stitched into the genome of C3H mice, <i>B. burgdorferi </i>infection no longer caused joint inflammation.<br />
<br />
Does the same process occur in humans with Lyme
arthritis? One hint that β-glucuronidase influences the course of Lyme
arthritis is the finding from other labs that found that the concentration of the enzyme in joint fluid is <a href="http://dx.doi.org/10.1080/00365540903036220" target="_blank">higher in patients with Lyme arthritis</a> than it is in healthy
uninfected individuals, although how the high enzyme levels are mechanistically linked
to arthritis remains unexplained.<br />
<br />
So how does β-glucuronidase deficiency lead to severe Lyme arthritis? One possibility raised by the authors is that GAG fragments worsen tissue inflammation by stimulating Toll-like receptors, as shown in other studies (see <a href="http://dx.doi.org/10.1038/nm1315" target="_blank">this paper</a> for an example).<br />
<br />
The findings may also tell us something about rheumatoid arthritis (RA). The B6 strain ends up with a form of RA following injection with certain autoantibodies. One of Weis' mouse crosses generated a B6 strain with its <i>Gusb</i> gene and flanking regions swapped for the same region of the C3H strain. This strain developed severe arthritis when injected with the same autoantibodies and when infected with <i>B. burgdorferi</i>. Therefore, the pathologic processes leading to Lyme arthritis and RA share common steps, at least in laboratory mice. In humans, RA patients, like those with Lyme arthritis, have high levels of β-glucuronidase levels in their joint fluid.<br />
<br />
The search for host factors affecting the development of Lyme arthritis goes on. Weis' group <a href="http://www.ncbi.nlm.nih.gov/pubmed/25378596" target="_blank">continue to identify genetic variants</a> responsible for severe Lyme arthritis.<br />
<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Journal+of+Clinical+Investigation&rft_id=info%3Apmid%2F24334460&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Lysosomal+%CE%B2-glucuronidase+regulates+Lyme+and+rheumatoid+arthritis+severity.&rft.issn=0021-9738&rft.date=2014&rft.volume=124&rft.issue=1&rft.spage=311&rft.epage=320&rft.artnum=&rft.au=Bramwell+KK&rft.au=Ma+Y&rft.au=Weis+JH&rft.au=Chen+X&rft.au=Zachary+JF&rft.au=Teuscher+C&rft.au=Weis+JJ&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology%2C+Genetics">Bramwell KK, Ma Y, Weis JH, Chen X, Zachary JF, Teuscher C, & Weis JJ (2014). Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity. <span style="font-style: italic;">The Journal of Clinical Investigation, 124</span> (1), 311-320 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24334460" rev="review">24334460</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Frontiers+in+Cellular+and+Infection+Microbiology&rft_id=info%3Apmid%2F24926442&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Forward+genetic+approaches+for+elucidation+of+novel+regulators+of+Lyme+arthritis+severity.&rft.issn=&rft.date=2014&rft.volume=4&rft.issue=&rft.spage=76&rft.epage=&rft.artnum=&rft.au=Bramwell+KK&rft.au=Teuscher+C&rft.au=Weis+JJ&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology%2C+Genetics">Bramwell KK, Teuscher C, & Weis JJ (2014). Forward genetic approaches for elucidation of novel regulators of Lyme arthritis severity. <span style="font-style: italic;">Frontiers in Cellular and Infection Microbiology, 4</span> PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24926442" rev="review">24926442</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Scandinavian+Journal+of+Infectious+Diseases&rft_id=info%3Apmid%2F19513935&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Activity+of+lysosomal+exoglycosidases+in+serum+and+synovial+fluid+in+patients+with+chronic+Lyme+and+rheumatoid+arthritis.&rft.issn=0036-5548&rft.date=2009&rft.volume=41&rft.issue=8&rft.spage=584&rft.epage=589&rft.artnum=&rft.au=Pancewicz+S&rft.au=Popko+J&rft.au=Rutkowski+R&rft.au=Kna%C5%9B+M&rft.au=Grygorczuk+S&rft.au=Guszczyn+T&rft.au=Bruczko+M&rft.au=Szajda+S&rft.au=Zajkowska+J&rft.au=Kondrusik+M&rft.au=Sierakowski+S&rft.au=Zwierz+K&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology%2C+Genetics">Pancewicz S, Popko J, Rutkowski R, Knaś M, Grygorczuk S, Guszczyn T, Bruczko M, Szajda S, Zajkowska J, Kondrusik M, Sierakowski S, & Zwierz K (2009). Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis. <span style="font-style: italic;">Scandinavian Journal of Infectious Diseases, 41</span> (8), 584-589 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/19513935" rev="review">19513935</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Nature+Medicine&rft_id=info%3Apmid%2F16244651&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Regulation+of+lung+injury+and+repair+by+Toll-like+receptors+and+hyaluronan.&rft.issn=1078-8956&rft.date=2005&rft.volume=11&rft.issue=11&rft.spage=1173&rft.epage=1179&rft.artnum=&rft.au=Jiang+D&rft.au=Liang+J&rft.au=Fan+J&rft.au=Yu+S&rft.au=Chen+S&rft.au=Luo+Y&rft.au=Prestwich+GD&rft.au=Mascarenhas+MM&rft.au=Garg+HG&rft.au=Quinn+DA&rft.au=Homer+RJ&rft.au=Goldstein+DR&rft.au=Bucala+R&rft.au=Lee+PJ&rft.au=Medzhitov+R&rft.au=Noble+PW&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology%2C+Genetics">Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, & Noble PW (2005). Regulation of lung injury and repair by Toll-like receptors and hyaluronan. <span style="font-style: italic;">Nature Medicine, 11</span> (11), 1173-1179 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16244651" rev="review">16244651</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-41626101605587052342014-03-10T10:55:00.001-07:002014-03-10T13:02:39.001-07:00Video microscopy of ticks acquiring the Lyme disease spirochete from miceThe bite of an infected <i>Ixodes</i> hard tick transmits the Lyme disease spirochete, <i>Borrelia burgdorferi</i>, to humans. Ticks acquire <i>B. burgdorferi</i> by feeding on <a href="https://en.wikipedia.org/wiki/Reservoir_host" target="_blank">reservoir hosts</a> colonized with the spirochete. Reservoir hosts include small mammals such as the white-footed mouse, the main reservoir of <i>B. burgdorferi</i> in the northeastern United States.<br />
<br />
A study that just came out in <cite>The Yale Journal of Biology and Medicine</cite> is accompanied by videos of <i>Borrelia burgdorferi</i> being transmitted between mouse and tick.<sup>1</sup> The authors prepared the mice by infecting them with <i>B. burgdorferi</i> genetically modified to express green fluorescent protein. After waiting two weeks to allow the spirochetes to disseminate, they placed one hungry <i>Ixodes scapularis</i> tick onto an ear of each infected mouse. <br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-B7oRyW916nA/Ux15UbZRxcI/AAAAAAAAAXg/yVo2j3GKk5w/s1600/Bockenstedt14-f1.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-B7oRyW916nA/Ux15UbZRxcI/AAAAAAAAAXg/yVo2j3GKk5w/s1600/Bockenstedt14-f1.png" height="320" width="317" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 from Bockenstedt et al., 2014<sup>1</sup>. A feeding tick (arrow) attached to the ear of a mouse. The tick is engorged with blood.</td></tr>
</tbody></table>
<br />
Nymphal ticks feed for an average of 2.5 to 8 days. The meal starts with the tick inserting its barbed feeding apparatus into the skin. (For a close-up view of this process, head over to the blog <a href="http://phenomena.nationalgeographic.com/2013/10/30/heres-what-happens-when-a-tick-bites-you/" target="_blank">Phenomena: Not Exactly Rocket Science</a>.) The tick releases saliva through the feeding canal into the skin. Tick saliva contains substances that damage host tissue surrounding the feeding apparatus and pharmacologic agents that inhibit clotting and engage the immune system. Intervals of salivation alternate with ingestion of blood, tissue fluid, and lymph that pool at the feeding site.<sup>2</sup> <br />
<br />
The authors examined the feeding site by two photon intravital microscopy to observe what was happening to the spirochetes. They saw spirochetes in the <a href="https://en.wikipedia.org/wiki/Dermis" target="_blank">dermis</a> moving towards the feeding apparatus and disappearing as they presumably got sucked into the feeding canal. One such spirochete is digitally colored in red in the video below, which was shot 48 hours into the blood meal. (One hour of video footage was compressed into 30 seconds.) The feeding apparatus is the green structure near the top of the viewing field. Previous studies have suggested that ticks acquire <i>B. burgdorferi</i> from skin, not from blood.<sup>3</sup> The videos from this study provide support for this notion, according to the authors.<br />
<br />
<div class="separator" style="clear: both; text-align: center;">
<iframe allowfullscreen='allowfullscreen' webkitallowfullscreen='webkitallowfullscreen' mozallowfullscreen='mozallowfullscreen' width='320' height='266' src='https://www.blogger.com/video.g?token=AD6v5dwco-t3-SrtboHJU8KIi0SUm_hVrq5B3Tl6bVJwRBNYKc3FihsYhwwhYzj84KRsHy6zV1OfcAEt0kHhmlBMPA' class='b-hbp-video b-uploaded' frameborder='0'></iframe></div>
<br />
Are the spirochetes mere passengers that get caught in the flow of fluid being drawn into the feeding canal, or are they active participants? The authors argue for an active role for the spirochetes:<br />
<blockquote>
Spirochete movement is unlikely to be due simply to the mechanical flux of tissue fluid as the tick feeds because close examination of individual spirochetes that move toward the <a href="https://en.wikipedia.org/wiki/Hypostome_%28tick%29" target="_blank">hypostome</a> reveals both the oscillating movements that we observe in the absence of tick feeding as well as directional translocation.</blockquote>
<br />
<br />
<b>References</b><br />
<br />
1. <span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Yale+Journal+of+Biology+and+Medicine&rft_id=info%3Apmid%2F24600332&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=What+ticks+do+under+your+skin%3A+two-photon+intravital+imaging+of+Ixodes+scapularis+feeding+in+the+presence+of+the+Lyme+disease+spirochete.&rft.issn=0044-0086&rft.date=2014&rft.volume=87&rft.issue=1&rft.spage=3&rft.epage=13&rft.artnum=&rft.au=Bockenstedt+LK&rft.au=Gonzalez+D&rft.au=Mao+J&rft.au=Li+M&rft.au=Belperron+AA&rft.au=Haberman+A&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Bockenstedt LK, Gonzalez D, Mao J, Li M, Belperron AA, & Haberman A (2014). What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the Lyme disease spirochete. <span style="font-style: italic;">The Yale Journal of Biology and Medicine, 87</span> (1), 3-13 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/24600332" rev="review">24600332</a></span><br />
<br />
2. <span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Infectious+Disease+Clinics+of+North+America&rft_id=info%3Apmid%2F18452797&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Biology+of+ticks.&rft.issn=0891-5520&rft.date=2008&rft.volume=22&rft.issue=2&rft.spage=195&rft.epage=&rft.artnum=&rft.au=Anderson+JF&rft.au=Magnarelli+LA&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Anderson JF, & Magnarelli LA (2008). Biology of ticks. <span style="font-style: italic;">Infectious Disease Clinics of North America, 22</span> (2) PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/18452797" rev="review">18452797</a></span><br />
<br />
3. <span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=The+Journal+of+infectious+diseases&rft_id=info%3Apmid%2F2732513&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Ingestion+of+Lyme+disease+spirochetes+by+ticks+feeding+on+infected+hosts.&rft.issn=0022-1899&rft.date=1989&rft.volume=160&rft.issue=1&rft.spage=166&rft.epage=7&rft.artnum=&rft.au=Nakayama+Y&rft.au=Spielman+A&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Nakayama Y, & Spielman A (1989). Ingestion of Lyme disease spirochetes by ticks feeding on infected hosts. <span style="font-style: italic;">The Journal of infectious diseases, 160</span> (1), 166-7 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2732513" rev="review">2732513</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-77830543348752900672013-12-16T10:34:00.000-08:002013-12-16T10:34:01.831-08:00"...and a dog with lepto in its pee."I saw this video over at the <a href="http://www.wormsandgermsblog.com/2013/12/articles/miscellaneous/the-twelve-days-of-zoonoses/" target="_blank">Worms & Germs</a> blog. It's a new take on a popular Christmas carol. Enjoy!<br />
<br />
<iframe allowfullscreen="" frameborder="0" height="315" src="//www.youtube.com/embed/mCCF1xdpJ8w" width="560"></iframe>
Lyrics<br />
On the twelfth day of Christmas my true love gave to me,<br />
Twelve tubs of Purell,<br />
Eleven raccoon roundworms, <br />
Ten cats-a-scratching<br />
Nine hungry hookworms,<br />
Eight dogs-a-biting,<br />
Seven cats with ringworm,<br />
Six big fat dog ticks,<br />
<i>Five cats with fleas.</i><br />
Four rats with cowpox.<br />
Three tapeworms,<br />
Two toxic turtles,<br />
<i><b>And a dog with lepto in its pee.</b></i>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-51542781321249493462013-12-13T00:31:00.000-08:002013-12-13T00:31:53.115-08:00Escape of the Lyme disease spirochete from the bloodstream involves multiple adhesins and receptorsMicrobial pathogens that cause systemic infections often travel within the circulatory system to spread throughout the host. Eventually, these pathogens exit from the bloodstream to get to their target organ. The first step of exit, adherence to the inner surface of the vessel wall, is probably the most challenging one because the rapidly flowing blood shoots the microbes through the capillaries. A <a href="http://dx.doi.org/10.1016/j.tim.2013.06.005" target="_blank">recent review</a> described the process akin to "a spider trying to gain a foothold on the wall of a garden hose with the tap turned on full." Most <i>in vitro</i> studies of adherence involve placement of microbe-mammalian cell cocultures in a stationary incubator. These static conditions poorly reflect what microbes experience as they are carried throughout the circulatory system. A better way to study vascular adhesion is to watch the process occur within a live animal.<br />
<br />
A Canadian group has been doing just that. As I explained in <a href="http://spirochetesunwound.blogspot.com/2009/01/watch-videos-of-lyme-disease-spirochete.html" target="_blank">this post</a>, they used intravital microscopy to shoot videos of the Lyme disease spirochete, <i>Borrelia burgdorferi</i>, escaping from skin capillaries of living mice (see the earlier post to watch a few of the videos). The bacteria were genetically engineered to express green florescent protein so that they could be visualized with a fluorescence microscope.<br />
<br />
From analyzing the videos, the investigators concluded that escape occurs in a series of steps. The spirochete first "tethers" itself to the wall. Within a second (assuming it doesn't let go), the spirochete starts crawling ("dragging") along the inner wall of the capillary. The crawling spirochete eventually squeezes between the cells in the vessel wall (endothelial cells) to complete its escape.<br />
<br />
In a follow-up study (see <a href="http://spirochetesunwound.blogspot.com/2009/02/lyme-disease-spirochete-hijacks.html" target="_blank">this post</a>) they showed that expression of <i>bbk32</i>, encoding a <i>B. burgdorferi</i> surface protein, restored the ability of a highly-passaged, nonadherent <i>B. burgdorferi</i> strain to tether and drag along the vessel wall. BBK32 clings to fibronectin and glycosaminoglycans (GAGs) <i>in vitro</i>. The Canadian group found that fibronectin, which circulates in the bloodstream, and GAGs, which line the inner surface of capillaries, were necessary for <i>B. burgdorferi</i> to interact with the microvasculature. Fibronectin, by its ability to anchor itself to GAGs, may serve as a lifeline that allows bacteria with fibronectin-binding proteins to tether themselves to the vessel wall.<br />
<br />
Moriarty and colleagues conducted a third study to gain a better molecular understanding of BBK32's role in vascular adhesion. The study was published in <cite>Molecular Microbiology</cite> over a year ago (here's the <a href="http://dx.doi.org/10.1111/mmi.12045" target="_blank">link</a> to the study), but I still think it's worth going over today because of the significance of their findings, not only for Lyme disease but also for other diseases involving bloodstream dissemination of microbial pathogens.<br />
<br />
Since separate surfaces of the BBK32 protein are responsible for fibronectin and GAG binding, the investigators wanted to see if BBK32's binding activities were deployed sequentially to carry out tethering and dragging. Therefore, BBK32 variants defective in binding one or the other host protein were constructed by deleting small segments in each binding region. The mutant genes encoding the variants were then introduced on plasmids into the highly-passaged, nonadhesive <i>B. burgdorferi</i> strain that they used in their earlier study. The transformants expressing the BBK32 variants were injected into the bloodstream of mice, and skin capillaries were examined by intravital microscopy so that the investigators could count the tethering and dragging interactions like they did in their two earlier studies.<br />
<br />
<i>B. burgdorferi</i> expressing the BBK32 variant defective in fibronectin binding (Δ158-182 in panel A below) underwent fewer tethering interactions than spirochetes expressing full-length BBK32 (BBK32 FL). On the other hand, the BBK32 variant that bound GAG poorly (Δ45-68) mediated as many tethering interactions as wild-type BBK32. These results indicate that the first step of adherence to the microvasculature, tethering, involves BBK32 interaction with fibronectin. The BBK32 variant defective in GAG binding was impaired in promoting the second step of vascular adherence, dragging (panel B).<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://1.bp.blogspot.com/-eAs36jnXq2A/UqEzYFl4SHI/AAAAAAAAAWQ/xejT3-syCJQ/s1600/Moriarty12-f5ab.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://1.bp.blogspot.com/-eAs36jnXq2A/UqEzYFl4SHI/AAAAAAAAAWQ/xejT3-syCJQ/s1600/Moriarty12-f5ab.jpg" height="208" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5 from Moriarty <i>et al</i>., 2012. BBK32 Δ45-68 binds GAG poorly; BBK32 Δ158-182 binds fibornectin poorly.</td></tr>
</tbody></table>
Therefore, the exit pathway involving BBK32 goes as follows. <i>B. burgdorferi</i> first uses BBK32 to tether to fibronectin, which is anchored to the
vessel wall. For the second step, BBK32 uses a different surface to bind to GAGs, allowing the spirochete to make more extensive contacts with the wall, leading to dragging interactions along the inner surface of the capillary. Subsequent steps involving sequential contact of other bacterial factors with other host factors results in penetration of the spirochete through the vessel wall.<br />
<br />
BBK32 is clearly capable of mediating vascular adhesion of an otherwise nonadherent <i>B. burgdorferi</i> mutant lacking most of its other adhesins. However, to determine whether BBK32 really has a role in vascular adhesion of infectious <i>B. burgdorferi</i> requires a loss-of-function analysis, as opposed to the gain-of-function experiment just described. Therefore, the investigators started with an infectious <i>B. burgdorferi</i> strain and knocked out the <i>bbk32</i> gene. They injected the <i>bbk32</i> mutant and wild-type strains into the bloodstream of mice and again counted the number of tethering and dragging interactions in skin capillaries. Here's where the results get interesting. In comparison with the wild-type strain, they found that knocking out <i>bbk32</i> reduced the number of vascular interactions by only 20%, and this reduction wasn't even statistically significant. This result indicates that the contribution of BBK32 to vascular adhesion in skin capillaries is minor, at best. Another <i>B. burgdorferi</i> adhesin (or adhesins) is responsible for the majority of vascular interactions in this tissue.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-_MaLHOwyVso/UqE2xIEkriI/AAAAAAAAAWc/sazVOizmQWo/s1600/Moriarty12-f2b.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-_MaLHOwyVso/UqE2xIEkriI/AAAAAAAAAWc/sazVOizmQWo/s1600/Moriarty12-f2b.jpg" height="320" width="301" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2B from Moriarty <i>et al</i>., 2012. "Interactions" is the sum of tethering and dragging interactions counted in skin capillaries.</td></tr>
</tbody></table>
<br />
So where in the host does BBK32 function as a major adhesin? Since <i>B. burgdorferi</i> is known to colonize joint tissue, the investigators decided to train their microscope on the capillaries supplying blood to the joints. They saw that the spirochetes underwent the same tethering and dragging interactions that they observed in the skin. When they compared the <i>bbk32</i> mutant against the infectious strain, they found that BBK32 accounts for roughly half of the tethering and dragging interactions (infectious vs. <i>bbk32</i> KO in the graph below). Whatever adhesin or adhesins are responsible for the other 50% do not interact with GAGs since coninjection of large amounts of a fibronectin peptide that binds GAGs failed to reduce the number of early vascular interactions of the <i>bbk32</i> mutant (<i>bbk32</i> KO vs. <i>bbk32</i> KO + FN-C/H II).<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-fFl41Em-O-I/UqfeKfrP0oI/AAAAAAAAAWs/6S4yuWHvJkU/s1600/Moriarty12-f2c.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-fFl41Em-O-I/UqfeKfrP0oI/AAAAAAAAAWs/6S4yuWHvJkU/s1600/Moriarty12-f2c.jpg" height="308" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2C from Moriarty <i>et al</i>., 2012. "Interactions" is the sum of tethering and dragging interactions counted in joint capillaries.</td></tr>
</tbody></table>
<br />
Here's the bottom line:<br />
<ul>
<li>BBK32 is capable of mediating the first two steps of <i>B. burgdorferi</i> adhesion to the vasculature. These steps involve distinct surfaces of BBK32 undgoing sequential contacts with fibronectin and GAGs.</li>
<li>Whether BBK32 is actually involved in vascular adhesion depends on where the spirochete is located within the host. For example, BBK32 has a major role in vascular adhesion in the joint, whereas it has little or no role in the skin.</li>
<li>Clearly, there are other (currently undiscovered) bacterial adhesins and host receptors that promote vascular adhesion and escape.</li>
</ul>
<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Molecular+Microbiology&rft_id=info%3Apmid%2F23095033&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Vascular+binding+of+a+pathogen+under+shear+force+through+mechanistically+distinct+sequential+interactions+with+host+macromolecules.&rft.issn=0950-382X&rft.date=2012&rft.volume=86&rft.issue=5&rft.spage=1116&rft.epage=31&rft.artnum=&rft.au=Moriarty+TJ&rft.au=Shi+M&rft.au=Lin+YP&rft.au=Ebady+R&rft.au=Zhou+H&rft.au=Odisho+T&rft.au=Hardy+PO&rft.au=Salman-Dilgimen+A&rft.au=Wu+J&rft.au=Weening+EH&rft.au=Skare+JT&rft.au=Kubes+P&rft.au=Leong+J&rft.au=Chaconas+G&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Moriarty TJ, Shi M, Lin YP, Ebady R, Zhou H, Odisho T, Hardy PO, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, & Chaconas G (2012). Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules. <span style="font-style: italic;">Molecular Microbiology, 86</span> (5), 1116-31 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/23095033" rev="review">23095033</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Trends+in+Microbiology&rft_id=info%3Apmid%2F23876218&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Illuminating+the+roles+of+the+Borrelia+burgdorferi+adhesins.&rft.issn=0966-842X&rft.date=2013&rft.volume=21&rft.issue=8&rft.spage=372&rft.epage=9&rft.artnum=&rft.au=Coburn+J&rft.au=Leong+J&rft.au=Chaconas+G&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Coburn J, Leong J, & Chaconas G (2013). Illuminating the roles of the Borrelia burgdorferi adhesins. <span style="font-style: italic;">Trends in Microbiology, 21</span> (8), 372-9 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/23876218" rev="review">23876218</a></span><br />
<br />
<b>Related posts</b><br />
<br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2009/01/watch-videos-of-lyme-disease-spirochete.html" target="_blank">Watch videos of the Lyme disease spirochete escaping from the bloodstream of live mice!</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2009/02/lyme-disease-spirochete-hijacks.html" target="_blank">The Lyme disease spirochete hijacks fibronectin to escape from the bloodstream</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com1tag:blogger.com,1999:blog-1574140332407591967.post-32778395496827444132013-10-15T20:07:00.000-07:002013-10-15T20:07:26.036-07:00Towards sterilizing immunity against Leptospira with a DNA vaccineIn my previous post, I described the failure of researchers to come up with a conventional protein-based subunit vaccine that confers sterilizing immunity against leptospirosis. What I mean by "conventional" subunit vaccine is a mixture of purified recombinant <i>Leptospira</i> protein with an adjuvant (either aluminum hydroxide or Freund's). Although an antibody response was detected against most proteins tested, immunization failed to prevent kidney colonization in every case, including those animals that survived challenge with lethal strains of <i>Leptosipra</i>. It's becoming clear that the leptospirosis vaccine field must move beyond simple formulations of protein plus adjuvant if sterilizing immunity is desired.<br />
<br />
Several labs have explored more modern approaches to delivering leptospirosis vaccines. Odir Dellagostin's group down in Brazil tested the efficacy of <a href="https://en.wikipedia.org/wiki/DNA_vaccine" target="_blank">DNA vaccines</a> in protecting hamsters against leptospirosis, as described in <a href="http://dx.doi.org/10.1128/CVI.00601-12" target="_blank">this</a> paper in <cite>Clinical and Vaccine Immunology</cite>. Forster and colleagues targeted the <i>Leptospira interrogans</i> LigA and LigB proteins, which are surface proteins that <a href="http://spirochetesunwound.blogspot.com/2013/01/ligb-of-leptospira-interrogans-avoiding.html" target="_blank">disrupt (or exploit) multiple host functions</a>.<br />
<br />
The investigators cloned various fragments of the lengthy <i>ligA</i> and <i>ligB </i>genes downstream of the human cytomegalovirus promoter of the commercial expression plasmid pTARGET. They mixed the plasmid DNA with aluminum hydroxide adjuvant and injected the material into the muscle of the hind leg of hamsters. The animals were given a booster with the same material 21 days later. An IgG immune response was detected against four of the the five Lig protein fragments being tested (see figure below).<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-7O0TCZyqeVI/UloUYJRaKkI/AAAAAAAAAVs/DxZep4N0PT0/s1600/Forster13-f2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://3.bp.blogspot.com/-7O0TCZyqeVI/UloUYJRaKkI/AAAAAAAAAVs/DxZep4N0PT0/s1600/Forster13-f2.jpg" height="215" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2 from Forster<i> et al.</i>, 2013. Sera were drawn before immunization and after the first and second immunizations with pTARGET-based <i>lig</i> plasmid DNA. Purified recombinant protein encoded by each plasmid was used as antigen in ELISAs. Left, middle, and right bar for each DNA: before immunization, after first immunization, and after second immunization, respectively.</td></tr>
</tbody></table>
<br />
21 days after the boost, the animals were challenged with a lethal strain of <i>L. interrogans</i>. The survival curves are shown below. Note that animals immunized with the vector alone (small filled circles) were all dead by day 11.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-PdPI2d5f6Jg/UloWgUv2-gI/AAAAAAAAAV4/o-U6rlrAbCM/s1600/Forster13-f3.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://3.bp.blogspot.com/-PdPI2d5f6Jg/UloWgUv2-gI/AAAAAAAAAV4/o-U6rlrAbCM/s1600/Forster13-f3.jpg" height="190" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3 from Forster <i>et al.</i>, 2013.</td></tr>
</tbody></table>
<br />
Among the <i>lig</i> gene fragments, the one expressing the "LigBrep" fragment was effective, protecting five of the eight animals in the group (62.5%) from death. LigBrep comprises amino acid residues 1 through 628 of LigB, whose total length is 1891 residues. Although the survival rate is nothing to get excited over, what distinguishes the LigBrep DNA vaccine from the conventional subunit vaccines tested in earlier studies is that the kidneys from 4 of the 5 survivors were culture negative, indicating that sterilizing immunity was achieved in 80% of the animals that survived infection. <br />
<br />
Another notable outcome of the study was that protection was achieved even though the challenge strain and the vaccine's <i>lig</i> gene originated from different <i>Leptospira</i> serovars. One of the problems with killed whole-cell vaccines is that they only protect against <i>Leptospira</i> serovars present in the vaccine formulation because they target LPS, whose structure varies among different serovars. <i>Leptospira</i> proteins tend to be similar in amino acid sequence across different species and are therefore more attractive as vaccines. (The "killed-whole leptospires" control plotted in the graph above was generated from the challenge strain).<br />
<br />
So how does DNA vaccination induce sterilizing immunity against <i>Leptospira</i>? As always, more studies are needed to explore this issue, but I will go ahead and speculate. DNA vaccines that are administered by standard injection stimulate a Th1-biased immune response. Studies with cattle have suggested that vaccines must stimulate Th1 immunity to minimize kidney colonization by <i>Leptospira</i> (see <a href="http://dx.doi.org/10.1128/CVI.00288-10" target="_blank">this</a> study, for example). Moreover, an <a href="http://dx.doi.org/10.1016/j.vaccine.2007.10.052" target="_blank">earlier study</a> by Dellagostin's group demonstrated sterilizing immunity against <i>L. interrogans</i> in some animals immunized with a <i>Mycobacterium bovis</i> BCG strain that was engineered to express LipL32, the major outer membrane protein of <i>L. interrogans</i>. BCG also stimulates Th1 immunity.<br />
<br />
Why would a Th1 response be necessary for sterilizing immunity against <i>Leptospira</i>? Th1 cytokines help steer B cells into producing an IgG isotype that is strongly recognized by Fc receptor on phagocytes. Consequently, bacteria bound by these IgG molecules are engulfed by opsonophagocytosis. During <i>Leptospira</i> infections, opsonophagocytosis clears spirochetes from the circulation during the antibody response, raising the possibility that opsonophagocytosis also leads to sterilizing immunity by vaccines that induce production of the "right" IgG.<br />
<br />
Th1 cells are also necessary for cellular immunity, which enhances the killing functions of macrophages so that they can rid themselves of intracellular pathogens. <i>Leptospira</i> is considered to be an extracellular pathogen. Nevertheless, there may be a transient intracellular phase that is critical during infection. Although intracellular <i>Leptospira</i> has not been observed <i>in vivo</i>, <i>L. interrogans</i> is known to <a href="http://dx.doi.org/10.1111/j.1462-5822.2011.01660.x" target="_blank">survive and replicate in cultured macrophages</a>.<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+and+Vaccine+Immunology+%3A+CVI&rft_id=info%3Apmid%2F23486420&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=A+conserved+region+of+leptospiral+immunoglobulin-like+A+and+B+proteins+as+a+DNA+vaccine+elicits+a+prophylactic+immune+response+against+leptospirosis.&rft.issn=1556-6811&rft.date=2013&rft.volume=20&rft.issue=5&rft.spage=725&rft.epage=731&rft.artnum=&rft.au=Forster+KM&rft.au=Hartwig+DD&rft.au=Seixas+FK&rft.au=Bacelo+KL&rft.au=Amaral+M&rft.au=Hartleben+CP&rft.au=Dellagostin+OA&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Forster KM, Hartwig DD, Seixas FK, Bacelo KL, Amaral M, Hartleben CP, & Dellagostin OA (2013). A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis. <span style="font-style: italic;">Clinical and Vaccine Immunology : CVI, 20</span> (5), 725-731 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/23486420" rev="review">23486420</a></span>
<br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+and+vaccine+immunology+%3A+CVI&rft_id=info%3Apmid%2F21288995&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=A+Leptospira+borgpetersenii+serovar+Hardjo+vaccine+induces+a+Th1+response%2C+activates+NK+cells%2C+and+reduces+renal+colonization.&rft.issn=1556-6811&rft.date=2011&rft.volume=18&rft.issue=4&rft.spage=684&rft.epage=91&rft.artnum=&rft.au=Zuerner+RL&rft.au=Alt+DP&rft.au=Palmer+MV&rft.au=Thacker+TC&rft.au=Olsen+SC&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Zuerner RL, Alt DP, Palmer MV, Thacker TC, & Olsen SC (2011). A <i>Leptospira borgpetersenii</i> serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. <span style="font-style: italic;">Clinical and Vaccine Immunology : CVI, 18</span> (4), 684-91 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21288995" rev="review">21288995</a></span>
<br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Vaccine&rft_id=info%3Apmid%2F18063449&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Recombinant+Mycobacterium+bovis+BCG+expressing+the+LipL32+antigen+of+Leptospira+interrogans+protects+hamsters+from+challenge.&rft.issn=0264-410X&rft.date=2007&rft.volume=26&rft.issue=1&rft.spage=88&rft.epage=95&rft.artnum=&rft.au=Seixas+FK&rft.au=da+Silva+EF&rft.au=Hartwig+DD&rft.au=Cerqueira+GM&rft.au=Amaral+M&rft.au=Fagundes+MQ&rft.au=Dossa+RG&rft.au=Dellagostin+OA&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Seixas FK, da Silva EF, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, & Dellagostin OA (2007). Recombinant <i>Mycobacterium bovis</i> BCG expressing the LipL32 antigen of <i>Leptospira interrogans</i> protects hamsters from challenge. <span style="font-style: italic;">Vaccine, 26</span> (1), 88-95 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/18063449" rev="review">18063449</a></span>
<br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Cellular+microbiology&rft_id=info%3Apmid%2F21819516&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Characteristic+features+of+intracellular+pathogenic+Leptospira+in+infected+murine+macrophages.&rft.issn=1462-5814&rft.date=2011&rft.volume=13&rft.issue=11&rft.spage=1783&rft.epage=1192&rft.artnum=&rft.au=Toma+C&rft.au=Okura+N&rft.au=Takayama+C&rft.au=Suzuki+T&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Toma C, Okura N, Takayama C, & Suzuki T (2011). Characteristic features of intracellular pathogenic <i>Leptospira</i> in infected murine macrophages. <span style="font-style: italic;">Cellular microbiology, 13</span> (11), 1783-1192 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21819516" rev="review">21819516</a></span>
<br />
<br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2013/09/is-sterilizing-immunity-against.html" target="_blank">Is sterilizing immunity against <i>Leptospira</i> possible with protein subunit vaccines?</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2011/05/new-attenuated-leptospirosis-vaccine.html" target="_blank">A new attentuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of <i>Leptospira</i></a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-29024444894522425262013-09-15T16:59:00.000-07:002013-09-15T17:03:12.988-07:00Is sterilizing immunity against Leptospira possible with protein subunit vaccines?With complete bacterial genome sequences now available, "reverse vaccinology" can be conducted to identify proteins that can function as subunit vaccines. The "gene first" approach of reverse vaccinology relies upon computer analysis of the genome sequence to identify encoded proteins with features common to known surface-exposed and secreted bacterial proteins. The selected genes can then be cloned and expressed as recombinant proteins. The proteins, which may number in the hundreds, are then purified for vaccine testing in the animal model appropriate for the bacterial pathogen. Reverse vaccinology has been employed successfully to find protective protein antigens against <i>Neisseria meningitidis</i> serogroup B, <i>Streptococcus pneumoniae</i>, group B <i>Streptococcus</i>, <i>Bacillus anthracis</i>, <i>Porphyromonas gingivalis</i>, and other bacterial pathogens (reviewed in <a href="http://dx.doi.org/10.1016/j.vaccine.2009.01.072" target="_blank">this</a> paper).<br />
<br />
This approach sounds straightforward but in practice may not always lead to identification of effective subunit vaccines. An important study from Ben Adler's group down in Monash University illustrates the challenges of finding a subunit vaccine that prevents chronic <i>Leptospira</i> infections. They focused on serovar Hardjo, which causes chronic infections in cattle. They selected 263 Hardjo genes that were predicted to encode surface-exposed, secreted, or lipid-modified proteins. Among these they successfully cloned and expressed 223 genes as 238 protein antigens in <i>E. coli</i>. Some genes were expressed as two or more fragments because of their large size. 210 of the 238 (88%) aggregated into inclusion bodies during expression and had to be kept dissolved in urea during their purification. (Strangely, the urea was not removed by dialysis prior to immunization.) The 238 purified proteins were mixed with an aluminum hydroxide adjuvant and injected into hamsters. 169 of the 238 proteins (71%) generated an antibody response, yet none succeeded in preventing colonization of the kidneys following challenge with a Hardjo strain.<br />
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It's been hard enough to find leptospiral proteins that protect hamsters from lethal disease when tested as vaccines (see <a href="http://dx.doi.org/10.4161/hv.7.11.17944" target="_blank">this article</a> for a review), yet Murray and colleagues sought proteins that protected against <i>Leptospira</i> colonization, a more difficult endeavor that has never been achieved with subunit vaccines. The Hardjo strain they used easily colonizes the kidneys yet fails to produce any signs of disease in hamsters. Although several studies have demonstrated that certain versions of the LigA and LigB proteins, when administered as vaccines, protect hamsters and mice from being killed by lethal strains of <i>Leptospira</i>, survivors are left with infected kidneys. A vaccine that protects against disease or death but not infection may be adequate for humans, who eventually clear the spirochetes from their kidneys even following a natural infection (assuming the disease doesn't kill them). However, vaccinated cattle infected with Hardjo may not be able to clear the spirochetes and will continue to shed infectious <i>Leptospira</i> into the environment, placing the entire herd and the workers handling them at risk of infection. Hardjo infections generally don't cause signs of disease in cattle, but they can cause fetal death and drop in milk production in cows.<br />
<br />
The choice of adjuvant and destruction of protective conformational epitopes by urea are possible reasons for failure to find a protective antigen. On the other hand, perhaps a different method for delivery of protein antigens into animals should been considered. Stay tuned.<br />
<br />
<b>Featured paper</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Vaccine&rft_id=info%3Apmid%2F23176980&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Evaluation+of+238+antigens+of+Leptospira+borgpetersenii+serovar+Hardjo+for+protection+against+kidney+colonisation.&rft.issn=0264-410X&rft.date=2013&rft.volume=31&rft.issue=3&rft.spage=495&rft.epage=499&rft.artnum=&rft.au=Murray+GL&rft.au=Lo+M&rft.au=Bulach+DM&rft.au=Srikram+A&rft.au=Seemann+T&rft.au=Quinsey+NS&rft.au=Sermswan+RW&rft.au=Allen+A&rft.au=Adler+B&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Murray GL, Lo M, Bulach DM, Srikram A, Seemann T, Quinsey NS, Sermswan RW, Allen A, & Adler B (2013). Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation. <span style="font-style: italic;">Vaccine, 31</span> (3), 495-499 PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/23176980" rev="review">23176980</a></span>
<br />
<br />
<b>Helpful reviews</b><br />
<br />
Dellagostin, O.A., Grassmann, A.A., Hartwig, D.D., Felix, S.R., da Silva, E.F., & McBride, A.J.A. (November 2011). Recombinant vaccines against leptospirosis. <cite>Human Vaccines</cite> 7(11):1215-1224. DOI: <a href="http://dx.doi.org/10.4161/hv.7.11.17944" target="_blank">10.4161/hv.7.11.17944</a><br />
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Serruto, D., Serino, L., Masignani, V., & Pizza, M. (May 26, 2009). Genome-based approaches to develop vaccines against bacterial pathogens. <cite>Vaccine</cite> 27(25-26):3245-3250. DOI: <a href="http://dx.doi.org/10.1016/j.vaccine.2009.01.072" target="_blank">10.1016/j.vaccine.2009.01.072</a><br />
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-74041532290756455082013-03-13T10:39:00.000-07:002013-03-13T11:13:03.206-07:00Triggering OspC production in Borrelia burgdorferi during tick feeding: Is temperature the real signal?The <i>Ixodes</i> tick, the vector of the Lyme disease spirochete, goes months without a meal. During this time, the <i>Borrelia burgdorferi</i> spirochetes living in its midgut live quiet lives, <a href="http://spirochetesunwound.blogspot.com/2011/10/lyme-disease-spirochete-feasts-on-tick.html" target="_blank">sipping on the tick's antifreeze</a> to sustain themselves. When the tick finally takes a blood meal from a warm-blooded victim, <i>B. burgdorferi </i>responds by producing a number of new proteins, some of which are needed for transmission to and infection of the mammalian host. Among these proteins is the outer surface lipoprotein OspC, whose function involves capture of tick (see <a href="http://spirochetesunwound.blogspot.com/2010/03/fresh-approach-to-lyme-disease.html" target="_blank">this post</a>) and mammalian host proteins. How does <i>B. burgdorferi</i> know when to start making these critical proteins? The favored model has been that the the warmth of the blood entering the tick triggers <i>B. burgdorferi</i> to make these proteins. It's been known for almost two decades that <i>B. burgdorferi</i> growing in culture medium produces miniscule amounts of OspC at low temperatures (23º-24ºC) and larger amounts at higher temperatures (32º-37ºC), as shown in the figure below from the <a href="http://dx.doi.org/10.1073/pnas.92.7.2909" target="_blank">classic 1995 report</a> by Tom Schwan and colleagues.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://4.bp.blogspot.com/-O23Tansv4ak/UT4QhkDnZmI/AAAAAAAAAVQ/C1GV_Lj1zo0/s1600/Schwan95-f4.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://4.bp.blogspot.com/-O23Tansv4ak/UT4QhkDnZmI/AAAAAAAAAVQ/C1GV_Lj1zo0/s1600/Schwan95-f4.jpg" height="640" width="232" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 from <a href="http://dx.doi.org/10.1073/pnas.92.7.2909" target="_blank">Schwan et al., 1995</a>. <i>B. burgdorferi</i> incubated at 24ºC (lanes 2 and 6), transferred from 24ºC to 37ºC (lanes 3 and 7), incubated at 37ºC (lanes 4 and 8), or transferred from 37ºC to 24ºC (lanes 5 and 9). Panel A, SDS-PAGE gel stained for total proteins with Coomassie brilliant blue. Arrow marks location of OspC. Panel B, Western blot with flagellin antibody (Fla) and OspC antibody.</td></tr>
</tbody></table>
As reasonable as this model sounds, findings from a recent paper from Brian
Stevenson's group (<a href="http://dx.doi.org/10.1128/JB.01956-12" target="_blank">Jutras et al., 2012</a>) challenge the model. Although not emphasized in earlier papers, the authors noted that <i>B. burgdorferi</i> multiplies much more quickly at higher temperatures. In their hands, <i>B. burgdorferi</i> proliferated with a doubling time of 32 hours at 23ºC and 12 hours at 34ºC. As expected, their Western blots showed that more OspC was produced by the spirochetes growing at the higher
temperature. Members of the Erp family of surface proteins, whose levels also rise during tick feeding, were produced at higher levels at the higher temperature as well, as shown in earlier studies. The investigators devised an experiment to test whether <i>B. burgdorferi</i> could tie OspC and Erp expression to its growth rate instead of temperature.<br />
<br />
The standard culture medium for <i>Borrelia</i> is BSK-II with 6% rabbit serum, a complex nutrient-rich concoction. They made two new formulations of the culture medium to slow the growth rate:
(1) quarter strength BSK-II with the rabbit serum
concentration remaining at 6%; (2) full-strength BSK-II with the rabbit serum concentration reduced to 1.2%. Medium #1 slowed the doubling time at 34ºC to 40
hours, and medium #2 reduced it to 32 hours. Western blots of the
spirochetes harvested from both cultures revealed low levels of the OspC and
Erp proteins. When these spirochetes were inoculated into the standard culture medium (BSKII/6% rabbit serum) and incubated at 34ºC, high levels of the proteins
were again detected. Therefore, <i>B. burgdorferi</i> is capable of adjusting OspC and Erp expression by monitoring its growth rate, even if the surrounding temperature does not change.<br />
<br />
The final experiment from the study demonstrates that not even growth rate is the direct signal. The authors froze <i>B. burgdorferi</i> at -80ºC for at least a month and then inoculated the bacteria into standard culture medium for incubation at 23ºC. As a control, bacteria being maintained at 34ºC were also transferred to standard culture for incubation at 23ºC. Both cultures grew with the same doubling time. Nevertheless, the spirochetes that were revived from the frozen state produced more OspC and Erp proteins that those that were initially maintained at 34ºC.<br />
<br />
So what's the real cue? Going back to the natural life cycle of <i>B. burgdorferi</i>, the spirochetes living in the unfed tick's midgut do not really grow or divide. The metabolism of <i>B. burgdorferi</i> is slowed by the nutrient-poor conditions in the tick's midgut. When the tick finally takes a blood meal, the surge of nutrients entering the tick signals <i>B. burgdorferi</i> to rev up its metabolism, triggering production of OspC. This model would explain why the frozen spirochetes, whose metabolism was undoubtedly slowed, were able to produce large amounts of OspC and Erp proteins when inoculated into standard culture medium at 23ºC, the temperature usually associated with diminished production of the proteins. The challenge will be to figure out how <i>B. burgdorferi</i> is sensing its metabolic state at the molecular level.<br />
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<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Journal+of+Bacteriology&rft_id=info%3Adoi%2F10.1128%2FJB.01956-12&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Changes+in+bacterial+growth+rate+govern+expression+of+the+Borrelia+burgdorferi+OspC+and+Erp+infection-associated+surface+proteins.&rft.issn=0021-9193&rft.date=2012&rft.volume=195&rft.issue=4&rft.spage=757&rft.epage=764&rft.artnum=http%3A%2F%2Fjb.asm.org%2Fcgi%2Fdoi%2F10.1128%2FJB.01956-12&rft.au=Jutras%2C+B.L.&rft.au=Chenail%2C+A.M.&rft.au=Stevenson%2C+B.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Jutras, B.L., Chenail, A.M., & Stevenson, B. (2012). Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins. <span style="font-style: italic;">Journal of Bacteriology, 195</span> (4), 757-764 DOI: <a href="http://dx.doi.org/10.1128/JB.01956-12" rev="review">10.1128/JB.01956-12</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Proceedings+of+the+National+Academy+of+Sciences&rft_id=info%3Adoi%2F10.1073%2Fpnas.92.7.2909&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Induction+of+an+outer+surface+protein+on+Borrelia+burgdorferi+during+tick+feeding.&rft.issn=0027-8424&rft.date=1995&rft.volume=92&rft.issue=7&rft.spage=2909&rft.epage=2913&rft.artnum=http%3A%2F%2Fwww.pnas.org%2Fcgi%2Fdoi%2F10.1073%2Fpnas.92.7.2909&rft.au=Schwan%2C+T.G.&rft.au=Piesman%2C+J.&rft.au=Golde%2C+W.T.&rft.au=Dolan%2C+M.C.&rft.au=Rosa%2C+P.A.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., & Rosa, P.A. (1995). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. <span style="font-style: italic;">Proceedings of the National Academy of Sciences, 92</span> (7), 2909-2913 DOI: <a href="http://dx.doi.org/10.1073/pnas.92.7.2909" rev="review">10.1073/pnas.92.7.2909</a></span><br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2012/07/borrelia-burgdorferi-needs-alternative.html" target="_blank"><i>Borrelia burgdorferi</i> needs the alternative sigma factor RpoS to flee from the tick's midgut</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2011/10/lyme-disease-spirochete-feasts-on-tick.html" target="_blank">The Lyme disease spirochete feasts on tick antifreeze</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2010/03/fresh-approach-to-lyme-disease.html" target="_blank">A fresh approach towards a Lyme disease vaccine: targeting the tick</a></li>
</ul>
Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-52302156005283882952013-02-19T10:50:00.001-08:002013-02-19T10:50:41.465-08:00Is the major outer membrane lipoprotein LipL32 really exposed on the surface of Leptospira?Here's a study that may come as a surprise to those in the leptospirosis field. The outer membrane lipoprotein LipL32 is believed to be the dominant protein on the cell surface of pathogenic species of <i>Leptospira</i>. However, according to a <a href="http://dx.doi.org/10.1371/journal.pone.0051025" target="_blank">new <cite>PLoS One</cite> article</a> written by Pinne and Haake at UCLA, LipL32 may not be present on the surface at all. This is an important issue to get right because the <a href="http://dx.doi.org/10.1128/IAI.01639-07" target="_blank">function proposed for LipL32, attachment to the extracellular matrix during infection</a>, assumes that the lipoprotein is exposed on the surface of the spirochete. More importantly, a number of research groups have already committed a lot of time and resources towards generating LipL32-based vaccines, which in current formulations confer (at best) weak protection against leptospirosis in rodent models (see <a href="http://dx.doi.org/10.1016/j.vetmic.2009.03.012" target="_blank">this</a> review for a critical analysis of the vaccine studies).<br />
<br />
Pinne and Haake assessed surface exposure of LipL32 by two methods. The first involved adding <a href="http://en.wikipedia.org/wiki/Proteinase_K" target="_blank">proteinase K</a> to suspensions of <i>Leptospira</i> to digest proteins exposed on the surface of the spirochete. When they did this, they saw that the known surface-exposed proteins OmpL37 and OmpL47 were degraded. On the other hand, LipL32 didn't break down at all unless the spirochetes were first lysed by boiling them in a detergent (see Western blot below).<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-Rne10LPZXyM/URPkdvJFvlI/AAAAAAAAAUQ/0wzQ5J9mcBs/s1600/Pinne13-f1b.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-Rne10LPZXyM/URPkdvJFvlI/AAAAAAAAAUQ/0wzQ5J9mcBs/s1600/Pinne13-f1b.jpg" height="285" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1B from Pinne and Haake (2013). Increasing concentrations of proteinase K (up to 150 ug/ml) were added to suspensions (first five lanes) or lysates (last five lanes) of <i>L. interrogans</i>. Following incubation, LipL32 was examined in a Western blot. <a href="http://dx.doi.org/10.1371/journal.pone.0051025.g001" target="_blank">Source</a>.</td></tr>
</tbody></table>
<br />
They next added LipL32 antibodies to <i>Leptospira</i> to see if they bound to the surface of the spirochete. They did not, providing additional evidence that LipL32 was not exposed on the cell surface. The authors tested LipL32 antibodies from different sources in an attempt to rule out the possibility that failure of antibody binding was due to the surface-exposed portion of LipL32 not being antigenic. LipL32 antiserum raised in rabbits, monoclonal LipL32 antibodies raised in mice, and LipL32 antibodies purified from the sera of leptospirosis patients all failed to bind the surface of <i>Leptospira</i> unless the outer membrane was chemically (with methanol or EDTA) or physically disrupted. Note that antibodies raised against OmpL54, a known suface-exposed protein, reacted strongly with intact <i>Leptospira</i> (last pair of images below).<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-57Gx_fnc-CM/URPwfX5tdmI/AAAAAAAAAUg/g00rJQmp8pw/s1600/Pinne13-f3a.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://3.bp.blogspot.com/-57Gx_fnc-CM/URPwfX5tdmI/AAAAAAAAAUg/g00rJQmp8pw/s1600/Pinne13-f3a.jpg" height="640" width="224" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3A from Pinne and Haake (2013). Bound antibody was detected with a secondary fluorescent antibody (green). The spirochetes were also stained with <a href="http://en.wikipedia.org/wiki/DAPI" target="_blank">DAPI</a>, a penetrating dye that stains DNA (blue). Left column, intact <i>Leptospira</i>, right column, methanol-treated <i>Leptospira</i>. <a href="http://dx.doi.org/10.1371/journal.pone.0051025.g003" target="_blank">Source</a>.</td></tr>
</tbody></table>
<br />
In light of these results, the authors took another look at the <a href="http://iai.asm.org/content/73/8/4853.long" target="_blank">2005 study</a> by Cullen and coauthors, who claimed LipL32 was surface exposed. In contrast to Pinne and Haake, Cullen and colleagues detected binding of LipL32-specific antibodies to intact <i>Leptospira</i> in three different assays. However, Pinne and Haake point out that antibody binding in their assays was extremely weak when the abundance of LipL32 is considered. The most striking example was the immunoelectron microscopy image of <i>Leptospira</i> treated with gold-labeled LipL32 antibody (see next image). Yes, the surface ended up labeled, with a mean of 10.8 gold particles per spirochete cell. However, we now know that there are 38,000 copies of LipL32 in each bacterial cell, making LipL32 the most abundant protein of <i>L. interrogans</i> (see <a href="http://spirochetesunwound.blogspot.com/2009/08/protein-census-in-leptospira.html" target="_blank">this blog post</a> about the <i>Leptospira</i> protein census). If LipL32 were really surface exposed, the surface of the spirochete should have been packed with gold particles.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-4nMLmhO2p5s/URPp6ozUGcI/AAAAAAAAAUY/ivMCn8r_b1I/s1600/Cullen05-f5.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://2.bp.blogspot.com/-4nMLmhO2p5s/URPp6ozUGcI/AAAAAAAAAUY/ivMCn8r_b1I/s1600/Cullen05-f5.jpg" height="400" width="385" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 5 from Cullen <i>et al.</i> (2005).</td></tr>
</tbody></table>
<br />
Cullen and colleagues also mixed suspensions of <i>Leptospira</i> with a biotin probe that reacts with primary amines (mostly on lysine side chains). The probe should have reacted solely with surface-exposed proteins since it's unable to penetrate the outer lipid bilayer and is assumed to be too large to diffuse through outer membrane <a href="http://en.wikipedia.org/wiki/Porin_%28protein%29" target="_blank">porins</a>. Biotinylated proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry (see next figure). Although LipL32 was one of the few proteins labeled with biotin, it's hard to make a firm conclusion about its surface exposure because proteins known to be located underneath the outer membrane, FlaB1 (a flagellar protein) and GroEL (a cytoplasmic heat shock protein), were also labeled with biotin. It's possible that LipL32 was labeled despite being located underneath the surface because the membrane was damaged while the spirochetes were being harvested for the experiment.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://4.bp.blogspot.com/-tsKO29OyhbM/UR6CoR65kJI/AAAAAAAAAVA/JU7lawPF6Rw/s1600/Cullen05-f2.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://4.bp.blogspot.com/-tsKO29OyhbM/UR6CoR65kJI/AAAAAAAAAVA/JU7lawPF6Rw/s1600/Cullen05-f2.png" height="245" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Modified from Figure 2 of Cullen <i>et al.</i>, 2005. Biotinylated proteins were separated by two-dimnesional electrophoresis. Spots were removed and analyzed by mass spectrometry to identify proteins. Multiple spots for each protein in the result of members of the population of each protein reacting with different numbers of biotin molecules. "LipL32.16" was generated by proteolysis of LipL32.</td></tr>
</tbody></table>
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<div class="separator" style="clear: both; text-align: center;">
</div>
Based on the intense labeling of LipL32 with biotin, Cullen and coauthors declared LipL32 the most abundant protein on the cell surface. They speculated that LipL32 is
poorly accessible to large molecules such as antibodies and proteases
(which in their study failed to digest <i>any</i> protein when added to intact <i>Leptospira</i>) because the LPS side chains act as a "rainforest canopy" that can be penetrated only by smaller molecules such as biotin. This is a reasonable supposition because LipL32 is up to <a href="http://dx.doi.org/10.1074/jbc.M109.006320" target="_blank">60Å in length</a>, whereas the distance between the outer membrane and the surface of the LPS layer is 92Å, according to a <a href="http://dx.doi.org/10.1128/JB.06474-11" target="_blank">cryoelectron microscopy study of <i>L. interrogans</i></a>. On the other hand, Pinne and Haake concluded that LipL32 is entirely or almost entirely subsurface since their assays failed to detect even a hint of the lipoprotein on the surface of <i>Leptospira</i>. They maintain that the reactivity of surface probes with LipL32 observed by Cullen and colleagues was an artifact generated by the presence of damaged spirochetes in their assays and the massive copy number of LipL32. <br />
<br />
The results from Pinne and Haake's study do not rule out the "rainforest canopy" model since they did not test smaller surface probes that could penetrate into the LPS side chain layer. Additional studies are needed to pin down the location of LipL32 relative to the surface of <i>Leptospira</i>.<br />
<b><br /></b>
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+ONE&rft_id=info%3Adoi%2F10.1371%2Fjournal.pone.0051025&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=LipL32+is+a+subsurface+lipoprotein+of+Leptospira+interrogans%3A+Presentation+of+new+data+and+reevaluation+of+previous+studies.&rft.issn=1932-6203&rft.date=2013&rft.volume=8&rft.issue=1&rft.spage=0&rft.epage=&rft.artnum=http%3A%2F%2Fdx.plos.org%2F10.1371%2Fjournal.pone.0051025&rft.au=Pinne%2C+M.&rft.au=Haake%2C+D.A.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Pinne, M., & Haake, D.A. (2013). LipL32 is a subsurface lipoprotein of Leptospira interrogans: Presentation of new data and reevaluation of previous studies. <span style="font-style: italic;">PLoS ONE, 8</span> (1) DOI: <a rev="review" href="http://dx.doi.org/10.1371/journal.pone.0051025">10.1371/journal.pone.0051025</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Infection+and+Immunity&rft_id=info%3Adoi%2F10.1128%2FIAI.73.8.4853-4863.2005&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Surfaceome+of+Leptospira+spp.&rft.issn=0019-9567&rft.date=2005&rft.volume=73&rft.issue=8&rft.spage=4853&rft.epage=4863&rft.artnum=http%3A%2F%2Fiai.asm.org%2Fcgi%2Fdoi%2F10.1128%2FIAI.73.8.4853-4863.2005&rft.au=Cullen%2C+P.A.&rft.au=Xu%2C+X.&rft.au=Matsunaga%2C+J.&rft.au=Sanchez%2C+Y.&rft.au=Ko%2C+A.I.&rft.au=Haake%2C+D.A.&rft.au=Adler%2C+B.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake, D.A., & Adler, B. (2005). Surfaceome of Leptospira spp. <span style="font-style: italic;">Infection and Immunity, 73</span> (8), 4853-4863 DOI: <a rev="review" href="http://dx.doi.org/10.1128/IAI.73.8.4853-4863.2005">10.1128/IAI.73.8.4853-4863.2005</a></span><br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2009/08/protein-census-in-leptospira.html" target="_blank">Protein census of <i>Leptospira interrogans</i></a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2010/08/major-outer-membrane-protein-of.html" target="_blank">The major outer membrane protein of <i>Leptospira interrogans</i>: Not essential for infection?</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-74145981804234167452013-02-04T13:18:00.001-08:002013-02-04T13:18:47.124-08:00An autoantigen targeted during Lyme arthritisInfection by the spirochete <i>Borrelia burgdorferi</i>, if left untreated, can lead to a form of Lyme disease called Lyme arthritis. About 10% of Lyme arthritis patients end up with a chronic form that doesn't go away with antibiotic treatment. Allen Steere's group has long suspected that the antibiotic-refractory form of Lyme arthritis involves an autoimmune process. This notion seems reasonable since those with antibiotic-refractory Lyme arthritis tend to have certain forms of the <a href="http://en.wikipedia.org/wiki/HLA-DR" target="_blank">HLA-DR</a> gene that are also common among those afflicted with the autoimmune disease <a href="http://en.wikipedia.org/wiki/Rheumatoid_arthritis" target="_blank">rheumatoid arthritis</a>.<br />
<br />
Several groups have been searching for autoantigens (self antigens) that could drive the joint inflammation seen in Lyme arthritis patients. Several candidate protein autoantigens were identified based on their short sequence similarities (<a href="http://en.wikipedia.org/wiki/Molecular_mimicry" target="_blank">molecular mimicry</a>) to a T-cell or antibody <a href="http://en.wikipedia.org/wiki/Epitope" target="_blank">epitopes</a> in the <i>B. burgdorferi</i> OspA protein, which is targeted by the immune system in many Lyme arthritis patients, especially those with the antibiotic-refractory form. However, further studies demonstrated that none of these autoantigens were likely to stimulate a sufficiently robust T-cell or antibody response that could account for the prolonged joint swelling experienced by patients with antibiotic-refractory Lyme disease (see this excellent <a href="http://dx.doi.org/10.1093/cid/ciq117" target="_blank">review article</a> for the complete story). Therefore, an additional approach is needed to identify additional autoantigen candidates, an approach that does not assume that molecular mimicry underlies antibiotic-refractory Lyme arthritis.<br />
<br />
An unbiased approach for finding autoantigens is to gather all of the different self-peptides being displayed by the HLA-DR molecules in the synovial tissue of the swollen joint and then figure out which of these peptides are capable of stimulating T cells. At one time this approach wasn't possible since the individual peptides presented by HLA-DR molecules are found in such tiny amounts in human tissues, but the sensitivity of today's liquid chromatography/tandem mass spectrometry systems have improved to the point where many of the peptides can now be sorted and sequenced. <br />
<br />
Steere's <a href="http://dx.doi.org/10.1002/art.37732" target="_blank">new study</a>, which appeared in January's print issue of <cite>Arthritis and Rheumatism</cite>, was conducted in collaboration with Catherine Costello's group in Boston University. The study was a follow-up to an <a href="http://dx.doi.org/10.1074/mcp.M110.002477" target="_blank">earlier one</a> published two years ago. The authors extracted the inflamed synovial tissue from the swollen knee of a 12 year old boy suffering from antibiotic-refractory Lyme arthritis (see picture below). He had gone through three months of antibiotic therapy a year prior to the procedure. The tissue was culture and PCR negative. When the patient's HLA-DR genes were examined, he turned out to have a copy of the
DRB1*0101 allele, one of the HLA-DRB gene variants that places individuals at a higher risk for antibiotic-refractory Lyme
arthritis.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://1.bp.blogspot.com/-NaUDeQZgKVA/UQXxLMPMajI/AAAAAAAAAUA/KH_D2hsLIVY/s1600/Drouin13-f1a.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://1.bp.blogspot.com/-NaUDeQZgKVA/UQXxLMPMajI/AAAAAAAAAUA/KH_D2hsLIVY/s1600/Drouin13-f1a.jpg" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1A from Drouin <i>et al</i>., 2013</td></tr>
</tbody></table>
<br />
The boy's synovial tissue was ground up, and HLA-DR-specific antibodies were used to capture the HLA-DR molecules with their bound peptides. The peptides were then analyzed by liquid chromatography/tandem mass spectrometry. The authors identified 120 different self-peptides from this analysis. When each peptide was chemically synthesized and mixed with the boy's blood mononuclear cells, one peptide turned out to stimulate proliferation of his T cells. This peptide came from a human protein called endothelial cell growth factor, or ECGF.<br />
<br />
What's the function of ECGF? The protein stimulates <a href="http://en.wikipedia.org/wiki/Angiogenesis" target="_blank">angiogenesis</a>,
the sprouting of new blood vessels from pre-existing ones.
Angiogenesis is a general feature of inflammatory arthritis, including
Lyme arthritis and rheumatoid arthritis. <br />
<br />
The authors went on to examine the T- and B-cell responses to ECGF in other Lyme arthritis patients. The T-cell response was determined by measuring the amount of interferon-γ secreted by the patients' blood mononuclear cells upon exposure to ECGF <i>in vitro</i>. In antibiotic-refractory patients, the T-cell response was observed in 38% (14/37) subjects against 30% (8/27) among Lyme arthritis patients who responded to antibiotics. The difference between the two groups was not statistically significant. The B-cell (antibody) response was examined by ELISA in a larger group of patients. 17% (19/109) of antibiotic-refractory patients and 8% (6/77) of antibiotic-responsive patients had an IgG antibody response against ECGF that was higher than among healthy controls, yet the difference between the antibiotic-refractory and -responsive groups again was not statistically significant (<i>P</i> = 0.09). So a link between an autoimmune response to ECGF and antibiotic-refractory arthritis was not clear-cut. However, in support of a link, the authors mentioned that almost all of the Lyme arthritis
patients with a T-cell response to ECGF (20/21, 98%) had one of the
HLA-DR alleles known to be a risk factor for antibiotic-refractory
arthritis.<br />
<br />
The authors also looked at the levels of ECGF in the
swollen joints of Lyme arthritis patients. Those with antibiotic-refractory Lyme arthritis had
much higher levels of ECGF in their joint fluid (mean 448 ng/ml, 37
subjects) than those whose arthritis responded to antibiotic treatment
(mean 154 ng/ml, 19 subjects, <i>P</i> < 0.0001)<br />
<br />
Further evidence for a link between an immune response to ECGF and chronic Lyme arthritis came from a group of untreated Lyme disease patients who were followed in the late 1970s, before the cause of Lyme disease was known. Sera from sequential bleeds were still available from many of these patients. If an autoimmune process involving ECGF was responsible for the disease, then the immune response to the autoantigen should have appeared before the disease symptoms. This turned out to be the case. Six of the seven Lyme arthritis patients who had antibodies against ECGF developed the antibody response before their joints swelled up. The duration of the arthritis attack was longer in Lyme arthritis patients with an immune response to ECGF, lasting a median of 67 weeks in the seven patients with an ECGF antibody response and only 17 weeks in the 20 Lyme arthritis lacking the response (<i>P</i> = 0.004).<br />
<br />
Steere's paper proposes that the immune response to ECGF leads to a persisting, autoimmune form of arthritis in those who have a high level of ECGF in their joint fluid. In those patients, T cells that recognize ECGF would be activated more easily because of the high levels of ECGF available for phagocytes to engulf, process, and display to the T cells. These events would lead to a chronic form of arthritis that would persist even when the spirochetes were cleared from the joints by the immune system or antibiotics. These patients also have a lot of ECGF in their synovial tissue. Antibody against ECGF could bind to the tissue and trigger attack by complement, contributing to the tissue damage.<br />
<br />
Molecular mimicry doesn't appear to be involved in triggering an immune response to ECGF. The authors were unable to identify any <i>B. burgdorferi</i> proteins that could cross-react with ECGF. <br />
<br />
The immune response to ECGF can't be the whole story since most patients with antibiotic-refractory Lyme arthritis don't generate a T-cell or antibody response to the protein. An autoimmune process in these other patients may involve other self-antigens waiting to be discovered. Other host and spirochete factors also influence the course of Lyme arthritis (see <a href="http://spirochetesunwound.blogspot.com/2009/12/genetics-of-both-host-and-pathogen.html" target="_blank">this post</a>, which gives the spirochete's point of view).<br />
<br />
<br />
<b>References</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Arthritis+%26+Rheumatism&rft_id=info%3Adoi%2F10.1002%2Fart.37732&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=A+novel+human+autoantigen%2C+endothelial+cell+growth+factor%2C+is+a+target+of+T+and+B+cell+responses+in+patients+with+Lyme+disease.&rft.issn=00043591&rft.date=2013&rft.volume=65&rft.issue=1&rft.spage=186&rft.epage=196&rft.artnum=http%3A%2F%2Fdoi.wiley.com%2F10.1002%2Fart.37732&rft.au=Drouin%2C+E.E.&rft.au=Seward%2C+R.J.&rft.au=Strle%2C+K.&rft.au=McHugh%2C+G.&rft.au=Katchar%2C+K.&rft.au=Londo%C3%B1o%2C+D.&rft.au=Yao%2C+C.&rft.au=Costello%2C+C.E.&rft.au=Steere%2C+A.C.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Drouin, E.E., Seward, R.J., Strle, K., McHugh, G., Katchar, K., Londoño, D., Yao, C., Costello, C.E., & Steere, A.C. (2013). A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease. <span style="font-style: italic;">Arthritis & Rheumatism, 65</span> (1), 186-196 DOI: <a href="http://dx.doi.org/10.1002/art.37732" rev="review">10.1002/art.37732</a></span><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Molecular+%26+Cellular+Proteomics&rft_id=info%3Adoi%2F10.1074%2Fmcp.M110.002477&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Peptides+presented+by+HLA-DR+molecules+in+synovia+of+patients+with+rheumatoid+arthritis+or+antibiotic-refractory+Lyme+arthritis.&rft.issn=1535-9476&rft.date=2010&rft.volume=10&rft.issue=3&rft.spage=0&rft.epage=0&rft.artnum=http%3A%2F%2Fwww.mcponline.org%2Fcgi%2Fdoi%2F10.1074%2Fmcp.M110.002477&rft.au=Seward%2C+R.J.&rft.au=Drouin%2C+E.E.&rft.au=Steere%2C+A.C.&rft.au=Costello%2C+C.E.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Seward, R.J., Drouin, E.E., Steere, A.C., & Costello, C.E. (2010). Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis. <span style="font-style: italic;">Molecular & Cellular Proteomics, 10</span> (3) DOI: <a href="http://dx.doi.org/10.1074/mcp.M110.002477" rev="review">10.1074/mcp.M110.002477</a></span>
<br />
<br />
<b>A helpful review</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Clinical+Infectious+Diseases&rft_id=info%3Adoi%2F10.1093%2Fcid%2Fciq117&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Relationship+between+immunity+to+Borrelia+burgdorferi+Outer-surface+protein+A+%28OspA%29+and+Lyme+arthritis.&rft.issn=1058-4838&rft.date=2011&rft.volume=52&rft.issue=Supplement+3&rft.spage=0&rft.epage=0&rft.artnum=http%3A%2F%2Fcid.oxfordjournals.org%2Flookup%2Fdoi%2F10.1093%2Fcid%2Fciq117&rft.au=Steere%2C+A.C.&rft.au=Drouin%2C+E.E.&rft.au=Glickstein%2C+L.J.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Steere, A.C., Drouin, E.E., & Glickstein, L.J. (2011). Relationship between immunity to Borrelia burgdorferi Outer-surface protein A (OspA) and Lyme arthritis. <span style="font-style: italic;">Clinical Infectious Diseases, 52</span> (Supplement 3) DOI: <a href="http://dx.doi.org/10.1093/cid/ciq117" rev="review">10.1093/cid/ciq117</a></span>
<br />
<br />
Related post<br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2009/12/genetics-of-both-host-and-pathogen.html" target="_blank">The genetics of both host and pathogen matter in antibiotic-refractory Lyme arthritis</a></li>
</ul>
Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-69918643831951310642013-01-14T11:42:00.001-08:002013-01-14T11:42:30.240-08:00LigB of Leptospira interrogans: Avoiding or exploiting complement?LigB has turned out to be a versatile surface protein for <i>Leptospira interrogans</i>. The protein is one of several that the spirochete uses to stick to the <a href="http://en.wikipedia.org/wiki/Extracellular_matrix" target="_blank">extracellular matrix</a>, a critical step in colonizing host tissues. In addition, LigB's ability to bind <a href="http://en.wikipedia.org/wiki/Fibrinogen" target="_blank">fibrinogen</a> may help <i>L. interrogans</i> spread within the host by <a href="http://dx.doi.org/10.1111/j.1365-2958.2010.07510.x" target="_blank">slowing clot formation</a>. According to separate studies from the U.S. and Brazil published last year, LigB also helps <i>L. interrogans</i> fend off attack by the host complement system.<br />
<br />
What's the evidence that LigB protects <i>Leptospira</i> from complement? <i>L. interrogans</i>, like many pathogens, survives even when complement is present. Its resistance to complement is reflected by its ability to survive in human serum, which contains all the complement components necessary to assemble the deadly <a href="http://en.wikipedia.org/wiki/Membrane_attack_complex" target="_blank">membrane attack complex</a> within exposed microbial membranes. On the other hand, the nonpathogen <i>Leptospira biflexa</i>, which lacks the <i>ligB</i> gene, is rapidly killed by human serum. The U.S. study demonstrated that transformation of <i>L. biflexa</i> with a plasmid expressing LigB allowed the nonpathogen to survive in human serum diluted to 5%, a concentration that easily killed <i>L. biflexa</i> harboring a similar plasmid lacking <i>ligB</i>.<br />
<br />
How exactly does LigB protect <i>Leptospira</i>
from the onslaught of complement? A defensive strategy deployed by many pathogens is to seize host <a href="http://en.wikipedia.org/wiki/Complement_control_protein" target="_blank">complement regulators</a>, which are present to prevent complement activation on host cells. The Brazilian study showed that LigB grabs several complement regulators that diminish the levels of the key complement proteins C3b and C4b on the bacterial surface. Two of these regulators are the <a href="http://en.wikipedia.org/wiki/Factor_H" target="_blank">factor H</a> protein and the C4-binding protein (C4BP). Both complement regulators break apart the <a href="http://en.wikipedia.org/wiki/C3_convertase" target="_blank">C3 convertases</a>, the enzyme complexes that generate C3b from C3. Factor H and C4BP also serve as cofactors for the protease <a href="http://en.wikipedia.org/wiki/Complement_factor_I" target="_blank">factor I</a>, which cleaves C3b and C4b into smaller pieces to prevent their assembly into the <a href="http://en.wikipedia.org/wiki/C5_convertase" target="_blank">C5 convertase</a>. The C5 convertase is what triggers assembly of the membrane attack complex. As expected, the investigators found that the breakdown of C3b and C4b by factor I in the presence of its complement regulators was accelerated when LigB was present.<br />
<br />
LigB is not the only <i>Leptospira</i> protein that captures complement regulators. The proteins <a href="http://iai.asm.org/content/74/5/2659.long" target="_blank">LenA</a> (originally called LfhA) and <a href="http://dx.doi.org/10.1128/IAI.00279-10" target="_blank">LcpA</a> bind factor H and C4BP, respectively. The importance of factor H in protecting <i>L. interrogans</i> can be seen in the bar graph below. Survival of <i>L. interrogans</i> was poor in serum lacking factor H. Addition of factor H to the serum up to the concentration found in blood (500 μg/ml) enhanced survival of the spirochete.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://1.bp.blogspot.com/-PhyXbSWY9Q0/UPN1vcOr8zI/AAAAAAAAATs/sbuFv777VGs/s1600/Castiblanco-Valencia13-f1.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="http://1.bp.blogspot.com/-PhyXbSWY9Q0/UPN1vcOr8zI/AAAAAAAAATs/sbuFv777VGs/s1600/Castiblanco-Valencia13-f1.jpg" height="288" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 1 from Castiblanco-Valencia et al. (2012). Survival of <i>L. interrogans</i> in factor H-depleted serum with 500 μg/ml factor H (FH) is set to 100%. </td></tr>
</tbody></table>
<br />
Surprisingly, the U.S. study revealed that a small segment of LigB grabbed C3b and C4b. Why would <i>L. interrogans</i> risk capturing complement proteins while simultaneously collecting complement regulators that inactivate those same proteins? Other pathogens do just fine capturing complement regulators without actively grabbing complement components. It's possible that C3b and C4b are inactivated more effectively by the complement regulators when all components are bound to LigB.<br />
<br />
There's another possibility that should be considered. What went unmentioned in both papers is that C3b and its cleavage products, which may remain attached to the bacterial surface, are <a href="http://en.wikipedia.org/wiki/Opsonin" target="_blank">opsonins</a> recognized by phagocytes aiming to grab and engulf microbial intruders. However, several intracellular pathogens use complement receptors as an entry point to invade macrophages. <i>L. interrogans</i> has been shown to survive within cultured macrophages and even remained intact within macrophages in a zebrafish model, as I've explained in <a href="http://spirochetesunwound.blogspot.com/2009/08/zebrafish-model-of-leptospirosis-wheres.html" target="_blank">another post</a>. Is it possible that <i>L. interrogans</i> uses C3b to grab and invade macrophages?<br />
<br />
<b>Featured papers</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Journal+of+Infectious+Diseases&rft_id=info%3Adoi%2F10.1093%2Finfdis%2Fjir875&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Leptospiral+immunoglobulin-like+proteins+interact+with+human+complement+regulators+factor+H%2C+FHL-1%2C+FHR-1%2C+and+C4BP.&rft.issn=0022-1899&rft.date=2012&rft.volume=205&rft.issue=6&rft.spage=995&rft.epage=1004&rft.artnum=http%3A%2F%2Fjid.oxfordjournals.org%2Flookup%2Fdoi%2F10.1093%2Finfdis%2Fjir875&rft.au=Castiblanco-Valencia+MM&rft.au=Fraga+TR&rft.au=da+Silva+LB&rft.au=Monaris+D&rft.au=Abreu+PAE&rft.au=Strobel+S&rft.au=Jozsi+M&rft.au=Isaac+L&rft.au=Barbosa+AS&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, & Barbosa AS (2012). Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. <span style="font-style: italic;">Journal of Infectious Diseases, 205</span> (6), 995-1004 DOI: <a rev="review" href="http://dx.doi.org/10.1093/infdis/jir875">10.1093/infdis/jir875</a></span>
<br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+ONE&rft_id=info%3Adoi%2F10.1371%2Fjournal.pone.0041566&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Multiple+activities+of+LigB+potentiate+virulence+of+Leptospira+interrogans%3A+Inhibition+of+alternative+and+classical+pathways+of+complement.&rft.issn=1932-6203&rft.date=2012&rft.volume=7&rft.issue=7&rft.spage=0&rft.epage=&rft.artnum=http%3A%2F%2Fdx.plos.org%2F10.1371%2Fjournal.pone.0041566&rft.au=Choy+H&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Choy H (2012). Multiple activities of LigB potentiate virulence of Leptospira interrogans: Inhibition of alternative and classical pathways of complement. <span style="font-style: italic;">PLoS ONE, 7</span> (7) DOI: <a rev="review" href="http://dx.doi.org/10.1371/journal.pone.0041566">10.1371/journal.pone.0041566</a></span>
<br />
<br />
<b>Other helpful papers</b><br />
<br />
Verma A, Hellwage J, Artiushin S, Zipfel PF, Kraiczy P, Timoney JF, and Stevenson B (March 2006). LfhA, a novel factor H-binding protein of <i>Leptospira interrogans</i>. <cite>Infection and Immunity</cite> 74(5):2659-2666. <a href="http://iai.asm.org/content/74/5/2659.long" target="_blank">Link</a><br />
<br />
Barbosa AS, Monaris D, Silva LB, Morais ZM, Vasconcellos SA, Cianciarullo AM, Isaac L, and Abreu PAE<span class="slug-doi"> (July 2010). Functional characterization of LcpA, a surface-exposed protein of <i>Leptospira</i> spp. that binds the human complement regulator C4BP. <cite>Infection and Immunity</cite> 78(7):3207-3216. DOI: <a href="http://10.1128/IAI.00279-10">10.1128/IAI.00279-10</a><a href="http://dx.doi.org/10.1128/IAI.00279-10" target="_blank">10.1128/IAI.00279-10</a></span><br />
<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2012/05/tick-protein-helps-lyme-disease.html" target="_blank">A tick protein helps the Lyme disease spirochete fight complement </a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2009/08/zebrafish-model-of-leptospirosis-wheres.html" target="_blank">Zebrafish model of leptospirosis: Where's the relevance?</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-47765388543387297992013-01-03T14:25:00.001-08:002013-01-03T14:35:30.627-08:00Spirochete research: 2012 in reviewHere are some of my favorite spirochete papers from 2012. Direct access to all research articles (some behind a paywall) is provided via the DOI links. Where present, the links above the citations lead to my blog posts about the studies.<br />
<br />
<h2>
PATHOGENESIS</h2>
Two distinct regions of the <i>Borrelia burgdorferi</i> BBK32 lipoprotein sequentially mediate binding to the vessel wall <i>in vivo</i> during escape of the spirochete from the bloodstream.<br />
<ul>
<li>Moriarty TJ, Shi , Lin Y-P, Ebady R,
Zhou H, Odisho T, Hardy P-O, Salman-Dilgimen A, Wu J, Weening EH, Skare
JT, Kubes P, Leong J, and Chaconas G (December 2012). Vascular binding
of a pathogen under shear force through mechanistically distinct
sequential interactions with host macromolecules. <cite>Molecular Microbiology</cite> 86(5):1116-1131. DOI: <a href="http://dx.doi.org/10.1111/mmi.12045" target="_blank">10.1111/mmi.12045</a></li>
</ul>
<br />
The <i>Leptospira interrogans</i> LigB protein protects the spirochete from complement by capturing complement regulatory proteins.<br />
<ul>
<li>Castiblanco-Valencia
MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M,
Isaac L, and Barbosa AS (March 15, 2012). Leptospiral
immunoglobulin-like proteins interact with human complement regulators
factor H, FHL-1, FHR-1, and C4BP. <cite>The Journal of Infectious Diseases</cite> 205(6):995-1004. DOI: <a href="http://dx.doi.org/10.1093/infdis/jir875" target="_blank">10.1093/infdis/jir875</a></li>
<li>Choy HA (July 2012). Multiple activities of LigB potentiate virulence of <i>Leptospira interrogans</i>:
inhibition of alternative and classical pathways of complement. <cite>PLoS One</cite> 7(7):e41566. DOI: <a href="http://dx.doi.org/10.1371/journal.pone.0041566" target="_blank">10.1371/journal.pone.0041566</a></li>
</ul>
<br />
A <i>B. burgdorferi</i> lipase with hemolytic activity <i>in vitro</i>:<br />
<ul>
<li>Shaw DK, Hyde JA, and Skare JT (January 2012). The BB0646 protein demonstrates lipase and haemolytic activity associated with <i>Borrelia burgdorferi</i>, the aetiological agent of Lyme disease. <cite>Molecular Microbiology</cite> 83(2):319-334. DOI: <a href="http://dx.doi.org/10.1111/j.1365-2958.2011.07932.x" target="_blank">10.1111/j.1365-2958.2011.07932.x</a> </li>
</ul>
<br />
<h2>
TRANSMISSION</h2>
<a href="http://spirochetesunwound.blogspot.com/2012/07/borrelia-burgdorferi-needs-alternative.html" target="_blank"><i>Borrelia burgdorferi</i> needs the alternative sigma factor RpoS to flee from the tick's midgut</a><br />
<ul>
<li>Dunham-Ems SM, Caimano MJ, Eggers CH, and Radolf JD
(February 2012). Borrelia burgdorferi requires the alternative sigma
factor RpoS for Dissemination within the vector during tick-to-mammal
transmission. <cite>PLoS Pathogens</cite> 8(2):e1002532. DOI: <a href="http://dx.doi.org/10.1371/journal.ppat.1002532" target="_blank">10.1371/journal.ppat.1002532</a></li>
</ul>
<br />
<h2>
MOTILITY</h2>
Video microscopy of <i>B. burgdorferi</i> swimming around in gelatin and mouse tissue:<br />
<ul>
<li>Harman MW, Dunham-Ems SM, Caimano MJ, Belperron AA, Bockenstedt LK, Fu HC, Radolf JD, and Wolgemuth CW (February 21, 2012). The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue. <cite>Proceedings of the National Academy of Sciences USA</cite> 109(8):3059-3064. DOI: <a href="http://dx.doi.org/10.1073/pnas.1114362109" target="_blank">10.1073/pnas.1114362109</a></li>
</ul>
<br />
<i>L. interrogans</i> sheath protein homologs that are not needed for flagellar sheath formation:<br />
<ul>
<li>Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, and Murray GA (June 2012). FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath. <cite>Infection and Immunity</cite> 80(6):2019-2025. DOI: <a href="http://dx.doi.org/10.1128/IAI.00131-12" target="_blank">10.1128/IAI.00131-12</a></li>
</ul>
<br />
<h2>
ULTRASTRUCTURE</h2>
A close look at the ultrastructure of <i>Leptospira</i> without the artifacts generated by conventional electron microscopy:<br />
<ul>
<li>Raddi G, Morado DR, Yan J,
Haake DA, Yang XF, and Liu J (March 2012). Three-dimensional
structures of pathogenic and saprophytic <i>Leptospira</i> species revealed by cryo-electron tomography. <cite>Journal of Bacteriology</cite> 194(6):1299-1306. DOI: <a href="http://dx.doi.org/10.1128/JB.06474-11" target="_blank">10.1128/JB.06474-11</a></li>
</ul>
<br />
<h2>
METAL TOXICITY</h2>
<i>B. burgdorferi</i> BicA, a protein that protects the spirochete from the toxic effects of copper and iron:<br />
<ul>
<li>Wang
P, Lutton A, Olesik J, Vali H, and Li X (December 2012). A novel iron-
and copper-binding protein in the Lyme disease spirochaete. <cite>Molecular Microbiology</cite> 86(6):1441-1451. DOI: <a href="http://dx.doi.org/10.1111/mmi.12068" target="_blank">10.1111/mmi.12068</a></li>
</ul>
<br />
<h2>
ANTIBIOTIC THERAPY</h2>
<a href="http://spirochetesunwound.blogspot.com/2012/11/inflammatory-spirochete-debris-left.html" target="_blank">Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease </a><br />
<ul>
<li>Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA (July 2, 2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy. The Journal of Clinical Investigation 122(7):2652-2660. DOI: <a href="http://dx.doi.org/10.1172/JCI58813" target="_blank">10.1172/JCI58813</a> </li>
</ul>
<br />
A critical analysis of a study that demonstrated persistence of <i>B. burgdorferi</i> in infected rhesus monkeys that were treated with antibiotics:<br />
<ul>
<li>Wormser
GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, and Shapiro ED (July
2012). Critical analysis of treatment trials of rhesus macaques
infected with <i>Borrelia burgdorferi</i> reveals important flaws in experimental design. <cite>Vector-borne and Zoonotic Diseases</cite> 12(7):535-538. DOI: <a href="http://dx.doi.org/10.1089/vbz.2012.1012" target="_blank">10.1089/vbz.<span class="highlight">2012</span>.1012</a></li>
<li>Embers ME, Barthold SW, Borda JT,
Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS,
Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, and
Philipp MT (January 2012). Persistence of <i>Borrelia burgdorferi</i> in rhesus macaques following antibiotic treatment of disseminated infection. <cite>PLoS One</cite> 7(1):e29914. DOI: <a href="http://dx.doi.org/10.1371/journal.pone.0029914" target="_blank">10.1371/journal.pone.0029914 </a></li>
</ul>
<br />
<div class="post-title entry-title">
<a href="http://spirochetesunwound.blogspot.com/2011/10/tale-of-two-more-studies-topical.html" target="_blank"><span style="font-size: small;">A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease</span></a></div>
<ul>
<li>Wormser
GP, Daniels TJ, Bittker S, Cooper D, Wang G, and Pavia CS (March 15,
2012). Failure of topical antibiotics to prevent disseminated <i>Borrelia burgdorferi</i> infection following a tick bite in C3H/HeJ mice. <cite>The Journal of Infectious Diseases</cite> 205(6):991-994. DOI: <a href="http://dx.doi.org/10.1093/infdis/jir382" target="_blank">10.1093/infdis/jir382</a></li>
</ul>
<br />
<h2>
DIAGNOSTICS</h2>
<a href="http://spirochetesunwound.blogspot.com/2012/07/not-so-golden-microscopic-agglutination.html" target="_blank">Not so golden? Microscopic agglutination test for diagnosis of leptospirosis</a><br />
<ul>
<li>Limmathurotsakul D, Turner EL, Wuthiekanun V,
Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ,
Cooper B, and Peacock SJ (August 1, 2012). Fool’s gold: Why imperfect
reference tests are undermining the evaluation of novel diagnostics: A
reevaluation of 5 diagnostic tests for leptospirosis. <cite>Clinical Infectious Diseases</cite> 55(3):322-331. DOI: <a href="http://dx.doi.org/10.1093/cid/cis403" target="_blank">10.1093/cid/cis403</a></li>
</ul>
<br />
<h2>
ECOLOGY</h2>
<a href="http://spirochetesunwound.blogspot.com/2012/05/do-nonspiral-spirochetes-help-clean-our.html" target="_blank">Do nonspiral spirochetes help clean our environment? </a><br />
<ul>
<li>Caro-Quintero A, Ritalahti KM, Cusick KD, Loffler FE, and Konstandtinidis KT (May/June 2012). The chimeric genome of <i>Sphaerochaeta</i>: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology. <cite>mBio</cite> 3(3):e00025-12. DOI: <a href="http://dx.doi.org/10.1128/mBio.00025-12" target="_blank">10.1128/mBio.00025-12</a></li>
<li>Ritalahti KM, Justicia-Leon SD, Cusick KD, Ramos-Hernandez N, Rubin M, Dornbush J, and Loffler FE (January 2012). <i>Sphaerochaeta globosa</i> gen. nov., sp. nov. and <i>Sphaerochaeta pleomorpha</i> sp. nov., free-living, spherical spirochetes. <cite>International Journal of Systematic and Evolutionary Microbiology</cite> 62(Pt 1):210-216. DOI: <a href="http://dx.doi.org/10.1099/ijs.0.023986-0" target="_blank">10.1099/ijs.0.023986-0</a></li>
</ul>
<h2>
</h2>
<h2>
BIOFILM</h2>
<a href="http://spirochetesunwound.blogspot.com/2012/12/biofilms-of-lyme-disease-spirochete.html" target="_blank">Biofilms of the Lyme disease spirochete</a><br />
<ul>
<li>Sapi
E, Bastian SL, Mpoy CM, Scott S, Rattelle A, Pabbati N, Poruri A,
Burugu D, Theophilus PAS, Pham TV, Data A, Dhaliwal NK, MacDonald A,
Rossi MJ, Sinha SK, and Luecke DF (October 2012). Characterization of
biofilm formation by <i>Borrelia burgdorferi in vitro</i>. <cite>PLoS One</cite> 7(10):e48277. DOI: <a href="http://dx.doi.org/10.1371/journal.pone.0048277" target="_blank">10.1371/journal.pone.0048277</a> </li>
</ul>
<h2>
</h2>
<h2>
HISTORY</h2>
<span style="font-size: small;"><a href="http://spirochetesunwound.blogspot.com/2012/06/looking-for-syphilis-spirochete-in.html" target="_blank">Looking <span style="font-size: small;">for t<span style="font-size: small;">he syphilis spirochete in ancient bones</span></span></a> </span><br />
<ul>
<li>Montiel R, Solorzano E, Diaz N, Alvarez-Sandoval BA, Gonzalez-Ruiz
M, Canadas MP, Simoes N, Isidro A, and Malgosa A (May 2012). Neonate
human remains: A window of opportunity to the molecular analysis of
syphilis. <cite>PLoS One</cite> 7(5):e36371. DOI: <a href="http://dx.doi.org/10.1371/journal.pone.0036371" target="_blank">10.1371/journal.pone.0036371</a></li>
</ul>
<br />
Presenting flawed studies directly to the public to bypass the scientific peer-review process:<br />
<ul>
<li>
<span style="font-size: small;">Armelagos GJ, Zuckerman MK, and Harper KN (March 2012).
The science behind pre-Columbian evidence of syphilis in Europe:
research by documentary. <cite>Evolutionary Anthropology</cite> 21(2):50-57. DOI: <a href="http://dx.doi.org/10.1002/evan.20340" target="_blank">10.1002/evan.20340</a></span></li>
</ul>
<br />
<h2>
REVIEWS</h2>
Here are two excellent review articles that appeared during the past year:<br />
<ul>
<li>Radolf JD, Caimano MJ, Stevenson B, and Hu LT (February 2012). Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. <cite>Nature Reviews Microbiology</cite> 10(2):87-99. DOI: <a href="http://dx.doi.org/10.1038/nrmicro2714" target="_blank">10.1038/nrmicro2714</a></li>
<li>Charon NW, Cockburn A, Li C, Liu J, Miller KA, Miller MR, Motaleb MA, and Wolgemuth CW (2012). The unique paradigm of spirochete motility and chemotaxis. <cite>Annual Reviews of Microbiology</cite> 66:349-370. DOI: <a href="http://dx.doi.org/10.1146/annurev-micro-092611-150145" target="_blank">10.1146/annurev-micro-092611-150145</a></li>
</ul>
<br />Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com1tag:blogger.com,1999:blog-1574140332407591967.post-72403343734760036762012-12-31T22:25:00.001-08:002013-01-03T17:18:23.618-08:00Biofilms of the Lyme disease spirocheteThanks to a <a href="http://dx.doi.org/10.1371/journal.pone.0048277" target="_blank">recent study</a> published in <cite>PLoS One</cite>, we now know that free-swimming <i>Borrelia burgdorferi</i> are able to organize themselves into a sedentary community called a <a href="http://en.wikipedia.org/wiki/Biofilm" target="_blank">biofilm</a>. This is not too surprising since most other bacteria are capable of the same feat when provided the opportunity. In fact, outside of the laboratory many bacteria, including those that live on and within us, spend much of their time within biofilms.<br />
<br />
Prior to the 1990s biofilms were thought to be blobs of goo containing bacteria randomly distributed throughout their sticky matrix. In reality, the bacteria and matrix are carefully organized into a complex three-dimensional structure. <i>B. burgdorferi</i> biofilms are no exception. The organization of <i>B. burgdorferi</i> is apparent even at the earliest stages of biofilm development. The images below show <i>B. burgdorferi</i> developing into a biofilm on a solid surface. Instead of randomly associating with each other, the spirochetes organize themselves into "nets" of the type you see hanging from basketball hoops. The spirochetes come together lengthwise to form the "strands" of the net. With time, the biofilm thickens as the bacteria form additional layers. Most of the
spaces in the net close up with the rest probably ending up forming a
network of channels. The remaining holes can be seen as pits along the
surface of the mature biofilm. The pits appear to be entry points for
the channels, which are thought to circulate nutrients to the members of the
community and remove waste products.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-XuhdfsnfR4o/UMqgkfSjKZI/AAAAAAAAASg/SWb3xf10VEg/s1600/Sapi12-f2.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="424" src="http://2.bp.blogspot.com/-XuhdfsnfR4o/UMqgkfSjKZI/AAAAAAAAASg/SWb3xf10VEg/s640/Sapi12-f2.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2 from Sapi <i>et al</i>. <a href="http://en.wikipedia.org/wiki/Atomic_force_microscopy" target="_blank">Atomic force microscopy</a> of a developing <i>B. burgdorferi</i> biofilm.</td></tr>
</tbody></table>
Stalks can also rise up from the
surface of microbial biofilms. One stalk can be seen in this "flyover" along the surface of a mature <i>B. burgdorferi</i> biofilm.<br />
<br />
<div class="separator" style="clear: both; text-align: center;">
<iframe allowfullscreen='allowfullscreen' webkitallowfullscreen='webkitallowfullscreen' mozallowfullscreen='mozallowfullscreen' width='320' height='266' src='https://www.blogger.com/video.g?token=AD6v5dygouZoivazV4BUa8ZD8VlOaHfAg0TLzeyOG-3RwYheNG9MjAI7pqo19KGPypONmNEK-uPEfaOtj--hLjDogA' class='b-hbp-video b-uploaded' frameborder='0'></iframe></div>
<div style="text-align: center;">
<span style="font-size: x-small;">Video S2 from Sapi <i>et al</i>. Composte image from atomic force microscopy.</span></div>
<br />
The matrix of <i>B. burgdorferi</i> biofilms includes DNA and an alginate-like substance bound to calcium. The images below capture what appears to be matrix being laid down at an early stage of biofilm formation. <br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://3.bp.blogspot.com/-vD7XN1vfKoM/UM560_wvGqI/AAAAAAAAAS0/N8V1YnU530k/s1600/Sapi12-f4.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="177" src="http://3.bp.blogspot.com/-vD7XN1vfKoM/UM560_wvGqI/AAAAAAAAAS0/N8V1YnU530k/s400/Sapi12-f4.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4 from Sapi <i>et al</i>. Atomic force microscopy of an aggregate of <i>B. burgdorferi</i> in an early stage of biofilm development. The matrix is colored blue in panel B.</td></tr>
</tbody></table>
<br />
What is the biological significance of <i>B. burgdorferi</i> biofilms? To answer this question, the authors will need to determine whether <i>B. burgdorferi</i> assembles into biofilms at some point during its life cycle, which involves stages in the tick and vertebrate host.<br />
<br />
<b>Reference</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=PLoS+ONE&rft_id=info%3Adoi%2F10.1371%2Fjournal.pone.0048277&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Characterization+of+biofilm+formation+by+Borrelia+burgdorferi+in+vitro.&rft.issn=1932-6203&rft.date=2012&rft.volume=7&rft.issue=10&rft.spage=0&rft.epage=&rft.artnum=http%3A%2F%2Fdx.plos.org%2F10.1371%2Fjournal.pone.0048277&rft.au=Sapi%2C+E.&rft.au=Bastian%2C+S.L.&rft.au=Mpoy%2C+C.M.&rft.au=Scott%2C+S.&rft.au=Rattelle%2C+A.&rft.au=Pabbati%2C+N.&rft.au=Poruri%2C+A.&rft.au=Burugu%2C+D.&rft.au=Theophilus%2C+P.A.S.&rft.au=Pham%2C+T.V.&rft.au=Datar%2C+A.&rft.au=Dhaliwal%2C+N.K.&rft.au=MacDonald%2C+A.&rft.au=Rossi%2C+M.J.&rft.au=Sinha%2C+S.K.&rft.au=Luecke%2C+D.F.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Sapi, E., Bastian, S.L., Mpoy, C.M., Scott, S., Rattelle, A., Pabbati, N., Poruri, A., Burugu, D., Theophilus, P.A.S., Pham, T.V., Datar, A., Dhaliwal, N.K., MacDonald, A., Rossi, M.J., Sinha, S.K., & Luecke, D.F. (2012). Characterization of biofilm formation by Borrelia burgdorferi in vitro. <span style="font-style: italic;">PLoS ONE, 7</span> (10) DOI: <a href="http://dx.doi.org/10.1371/journal.pone.0048277" rev="review">10.1371/journal.pone.0048277</a></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-92116582770501167152012-11-26T10:49:00.001-08:002012-12-05T08:48:06.868-08:00A post-Thanksgiving story of leptospirosisI'm about half way through <cite>1491</cite>, a book that gives readers a view of the Americas before Columbus showed up. It also describes the devastating impact that foreign infectious diseases had on the native population as Europeans explored the New World.<br />
<br />
One chapter tells the story of Tisquantum (Squanto), who lived in the village of Patuxet, one of the many Indian communities thriving along the coast of New England at the time. In 1614 Thomas Hunt, a British slave trader, kidnapped Tisquantum and other Indians and shipped them to Spain. Fortunately, Tisquantum was rescued by Spanish priests before he could be sold. After convincing the priests to let him return home, he left for London, where he learned English while staying at a shipbuilder's home, and eventually made his way back to North America. As he sailed down the New England shoreline in 1619 on a British ship, he realized that the world familiar to him had vanished. A mysterious disease had wiped out 90% of the population of coastal New England. When he arrived at his home village of Patuxet, he found it deserted. Tisquantum was soon captured and sent to Massasoit, the leader of the Wampanoag confederacy, which encompassed Patuxet. Massasoit did not trust Tisquantum because of his recent association with the British, yet he would later use him as a translator in a negotiation that turned out to be a pivotal event in American history.<br />
<div class="separator" style="clear: both; text-align: center;">
<a href="http://3.bp.blogspot.com/-GTbzWv2yM1s/ULLJbwIIimI/AAAAAAAAASA/vnWwtKBxayM/s1600/1491crop.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="200" src="http://3.bp.blogspot.com/-GTbzWv2yM1s/ULLJbwIIimI/AAAAAAAAASA/vnWwtKBxayM/s1600/1491crop.jpg" width="142" /></a></div>
<br />
The epidemic had been blamed at one time or another on smallpox, the plague, yellow fever, typhus, and hepatitis. As I've <a href="http://spirochetesunwound.blogspot.com/2010/02/did-spirochetes-kill-off-indians-of.html" target="_blank">mentioned before</a>, a <a href="http://dx.doi.org/10.3201/eid1602.090276" target="_blank">recent analysis</a> has added leptospirosis to the list of suspects. The symptoms and signs of leptospirosis match those reported from first-hand accounts of the mystery ailment. Here's a <a href="http://www.slate.com/articles/health_and_science/medical_examiner/2012/11/leptospirosis_and_pilgrims_the_wampanoag_may_have_been_killed_off_by_an.single.html" target="_blank">post</a> on the Slate website about the epidemic. I'm glad to see that the story is getting attention from popular news sites.<br />
<br />
Leptospirosis can be deadly, but could it account for the devastating 90% motality rate of the 1616-1619 epidemic? A hypervirulent strain of <i>Leptospira</i> or genetic susceptibility of the Indians could be an explanation. However, the authors of the study thought that the most critical factor was the Indian lifestyle, which brought them into repeated contact with <i>Leptospira</i> in the environment. The Europeans who fished nearby were spared because they did not engage in activities that exposed them to <i>Leptospira</i>. Therefore, only the Indians contracted the illness, according to the hypothesis.<br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="http://2.bp.blogspot.com/-_XhUu6Ou2fo/ULML_k8gPuI/AAAAAAAAASQ/WavwUtNGO_4/s1600/Marr10-f3.jpg" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="http://2.bp.blogspot.com/-_XhUu6Ou2fo/ULML_k8gPuI/AAAAAAAAASQ/WavwUtNGO_4/s1600/Marr10-f3.jpg" width="302" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3 from Marr and Cathey, 2010.</td></tr>
</tbody></table>
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Whatever its cause, it's hard to overstate the significance of the epidemic. Prior to 1616, the New England native communities traded with the Europeans and even welcomed them for brief stays. However, all attempts by the foreigners to establish permanent settlements were fiercely resisted. Coastal New England was well defended by the large native population. The Wampanoag confederacy became especially hostile towards the Europeans after having their citizens abducted.<br />
<br />
By the time the <i>Mayflower</i> landed in Patuxet (Plymouth) in December of 1620, the thinking of the Wampanoag had changed. Their depleted population was vulnerable to attack by their longtime enemies to the west, the Narragansett, who remained untouched by the epidemic. To forestall an attack, Massasoit felt that the best course of action was to form an alliance with the Pilgrims rather than expel them. In the spring of 1621, with Tisquantum serving as the translator, Massasoit arranged a peace treaty with the Pilgrims.<br />
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<b>Reference</b><br />
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<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft_id=info%3Adoi%2F10.3201%2Feid1602.090276&rft.atitle=New+hypothesis+for+cause+of+epidemic+among+Native+Americans%2C+New+England%2C+1616%E2%80%931619&rft.jtitle=Emerging+Infectious+Diseases&rft.artnum=http%3A%2F%2Fwwwnc.cdc.gov%2Feid%2Farticle%2F16%2F2%2F09-0276_article.htm&rft.volume=16&rft.issue=2&rft.issn=1080-6040&rft.spage=281&rft.epage=286&rft.date=2010&rfr_id=info%3Asid%2Fscienceseeker.org&rft.au=Marr+John+S.&rft.aulast=Marr&rft.aufirst=John+S.&rft.au=Cathey+John+T.&rft.aulast=Cathey&rft.aufirst=John+T.&rfs_dat=ss.included=1&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Marr J.S. & Cathey J.T. (2010). New hypothesis for cause of epidemic among Native Americans, New England, 1616–1619, <span style="font-style: italic;">Emerging Infectious Diseases, 16</span> (2) 281-286. DOI: <a href="http://dx.doi.org/10.3201%2Feid1602.090276" rel="author">10.3201/eid1602.090276</a></span><br />
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<b><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft_id=info%3Adoi%2F10.3201%2Feid1602.090276&rft.atitle=New+hypothesis+for+cause+of+epidemic+among+Native+Americans%2C+New+England%2C+1616%E2%80%931619&rft.jtitle=Emerging+Infectious+Diseases&rft.artnum=http%3A%2F%2Fwwwnc.cdc.gov%2Feid%2Farticle%2F16%2F2%2F09-0276_article.htm&rft.volume=16&rft.issue=2&rft.issn=1080-6040&rft.spage=281&rft.epage=286&rft.date=2010&rfr_id=info%3Asid%2Fscienceseeker.org&rft.au=Marr+John+S.&rft.aulast=Marr&rft.aufirst=John+S.&rft.au=Cathey+John+T.&rft.aulast=Cathey&rft.aufirst=John+T.&rfs_dat=ss.included=1&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Related post</span></b><br />
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<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2010/02/did-spirochetes-kill-off-indians-of.html" target="_blank"><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft_id=info%3Adoi%2F10.3201%2Feid1602.090276&rft.atitle=New+hypothesis+for+cause+of+epidemic+among+Native+Americans%2C+New+England%2C+1616%E2%80%931619&rft.jtitle=Emerging+Infectious+Diseases&rft.artnum=http%3A%2F%2Fwwwnc.cdc.gov%2Feid%2Farticle%2F16%2F2%2F09-0276_article.htm&rft.volume=16&rft.issue=2&rft.issn=1080-6040&rft.spage=281&rft.epage=286&rft.date=2010&rfr_id=info%3Asid%2Fscienceseeker.org&rft.au=Marr+John+S.&rft.aulast=Marr&rft.aufirst=John+S.&rft.au=Cathey+John+T.&rft.aulast=Cathey&rft.aufirst=John+T.&rfs_dat=ss.included=1&rfe_dat=bpr3.included=1;bpr3.tags=Biology">Did spirochetes kill off the Indians in Massachusetts before the Mayflower landed?</span></a></li>
</ul>
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft_id=info%3Adoi%2F10.3201%2Feid1602.090276&rft.atitle=New+hypothesis+for+cause+of+epidemic+among+Native+Americans%2C+New+England%2C+1616%E2%80%931619&rft.jtitle=Emerging+Infectious+Diseases&rft.artnum=http%3A%2F%2Fwwwnc.cdc.gov%2Feid%2Farticle%2F16%2F2%2F09-0276_article.htm&rft.volume=16&rft.issue=2&rft.issn=1080-6040&rft.spage=281&rft.epage=286&rft.date=2010&rfr_id=info%3Asid%2Fscienceseeker.org&rft.au=Marr+John+S.&rft.aulast=Marr&rft.aufirst=John+S.&rft.au=Cathey+John+T.&rft.aulast=Cathey&rft.aufirst=John+T.&rfs_dat=ss.included=1&rfe_dat=bpr3.included=1;bpr3.tags=Biology"><br /></span>Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com0tag:blogger.com,1999:blog-1574140332407591967.post-640421557355408762012-11-08T10:56:00.000-08:002012-11-08T10:56:37.595-08:00Inflammatory spirochete debris left behind following antibiotic treatment for Lyme diseaseAccording to the CDC, <a href="http://www.cdc.gov/lyme/postLDS/index.html" target="_blank">10-20%</a> of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain. The following hypotheses are often presented as possible reasons for the lingering symptoms: autoimmunity triggered by the infection, tissue damage inflicted by the spirochetes, and (depending on whom you ask) failure of antibiotics to kill all the spirochetes. A <a href="http://dx.doi.org/10.1172/JCI58813" target="_blank">new paper</a> from Linda Bockenstedt's group at Yale proposes that antibiotic treatment of disseminated <i>Borrelia burgdorferi</i> infection leaves behind inflammatory pieces of dead spirochetes that are responsible for the persisting symptoms.<br />
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Bockenstedt's group used the mouse model of Lyme disease for the study. To ensure that the tissues harbored enough <i>B. burgdorferi</i> spirochetes to be visible by intravital microscopy, the mice were genetically deficient in the intracellular signaling protein MyD88. MyD88 links the recognition of microbial parts by most <a href="http://en.wikipedia.org/wiki/Toll-like_receptor" target="_blank">Toll-like receptors</a> to activation of certain nuclear genes whose products are involved in the inflammatory process. Mice lacking MyD88 are unable to control the proliferation of a number of bacterial pathogens, including <i>B. burgdorferi</i>. The load of <i>B. burgdorferi</i> in tissues is about <a href="http://www.jimmunol.org/content/173/3/2003.long" target="_blank">100-fold higher in MyD88-deficient mice</a> than in mice with a complete immune system.<br />
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The spirochetes were genetically altered to express green fluorescent protein (GFP). The GFP<sup>+</sup> <i>B. burgdorferi</i> was introduced into the MyD88-deficient mice by tick inoculation. 21 days later some of the mice were treated for one month with <a href="http://en.wikipedia.org/wiki/Doxycycline" target="_blank">doxycycline</a>, one of the antibiotics used to treat Lyme disease in humans.<br />
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The researchers next peered into the thin layer of skin covering the ear by intravital microscopy. In the mice that were left untreated, they saw lots of spirochetes scurrying about in the dermis.<br />
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At the deepest depths of the dermis, they noticed immobile specks and patches of green material deposited near the cartilage. They also saw the deposits in the doxycycline-treated mice. The material was detected by immunofluorescence of ear sections with antibody against <i>B. burgdorferi</i> up to 10 weeks after antibiotic treatment was completed, indicating that the immune system was unable to clear the deposits.<br />
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There was no evidence that any spirochetes survived antibiotic treatment. The researchers did not see any motile spirochetes in the skin by intravital microscopy. In addition, tissues were culture negative, ticks that fed on the treated mice were culture negative (xenodiagnosis), and transplantation of skin from the treated mice failed to transmit the infection to recipient mice. Based on these results, the authors concluded that the deposits were remnants of dead spirochetes. As expected, untreated mice tested positive by these assays.<br />
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Since chronic infection can lead to Lyme arthritis, the investigators also examined the joints. In another set of mice, the infection was allowed to proceed for four months. The mice were then treated with the antibiotic <a href="http://en.wikipedia.org/wiki/Ceftriaxone" target="_blank">ceftriaxone</a> for 18 days. When the researchers looked in the joints by intravital microscopy, they again saw the green material (see figure below).<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="http://4.bp.blogspot.com/-ooteKzhzS1s/UIiWLi9HNAI/AAAAAAAAARo/79wtzKqiG0s/s1600/Bockenstedt12-f5.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="200" src="http://4.bp.blogspot.com/-ooteKzhzS1s/UIiWLi9HNAI/AAAAAAAAARo/79wtzKqiG0s/s1600/Bockenstedt12-f5.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fig. 5 from Bockenstedt <i>et al</i>. showing the surface of the patella where it meets the tendon (enthesis). Panel A, from mouse infected for 4 months, untreated. Panel B, from mouse infected for 4 months and then treated with ceftriaxone for 18 days. Scale bar, 30 µm.</td></tr>
</tbody></table>
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A critical issue to address is whether the amorphous material left behind following antibiotic treatment inflames the joints. The authors could not answer this question directly because of the limitations of the mouse model. Histopathology is unlikely reveal joint inflammation, even in the untreated animals, because laboratory mice do not reliably exhibit joint inflammation so late (4-5 months) during <i>B. burgdorferi</i> infection. Instead, the authors conducted a test tube experiment to see whether the deposits had inflammatory potential. They ground up joint tissue from antibiotic-treated mice in buffer and applied the homogenate to cultured mouse macrophages. The macrophages responded by producing TNF, a key cytokine that promotes inflammation. The more tissue that was added, the more TNF that was produced by the macrophages. In contrast, joint tissue from uninfected mice did not promote TNF production by the macrophages. Therefore, the deposits had the potential to spark inflammation, even after motile spirochetes were eliminated by antibiotics. The debris would continue to inflame the tissues even after antibiotics killed all live spirochetes, explaining why symptoms persist in ~10% of Lyme arthritis cases even after antibiotic treament.<br />
<br />
The relevance of the deposits to Lyme disease in humans
could be questioned because the MyD88-deficient mice did not have a
complete immune system. The authors addressed this concern in the
Discussion by mentioning a recent study that described <a href="http://dx.doi.org/10.1002/art.34383" target="_blank">a TLR1 variant</a>
linked to severe inflammation and treatment failure in Lyme arthritis
patients. Although the gene encoding MyD88 has never been examined in Lyme disease patients, it is conceivable that the TLR1
variant or different forms of other immune genes lead to deposits of <i>Borrelia</i> antigen in the joint and other host tissues.<br />
<br />
The authors also addressed the possibility that the deposits are really <a href="http://en.wikipedia.org/wiki/Biofilm" target="_blank">biofilms</a>, which generally resist killing by antibiotics. Biofilms are believed to be populated by persister cells, which are in a nondividing state that allows bacteria to tolerate antibiotics. According to the authors, if the deposits had harbored persister cells, those cells should have resumed growing when conditions became favorable for growth again. Because the skin and joints from the treated mice were culture negative and because the skin also tested negative by xenodiagnosis and transplantation assays, the authors quickly dismissed the biofilm hypothesis. <br />
<br />
Stricly speaking, the authors are correct. Persister cells should start multiplying again in fresh culture medium. However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see <a href="http://www.ncbi.nlm.nih.gov/pubmed/18049376" target="_blank">this review</a> for one example). Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris.<br />
<br />
Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients. Allen Steere's group <a href="http://dx.doi.org/10.1002/1529-0131(199912)42:12%3C2705::AID-ANR29%3E3.0.CO;2-H" target="_blank">failed to find such deposits</a> in pieces of <a href="http://en.wikipedia.org/wiki/Synovial_membrane" target="_blank">synovial membrane</a> removed from 26 patients with antibiotic-refractory Lyme arthritis. The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place. <br />
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<br />
<b>Featured paper</b><br />
<br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Journal+of+Clinical+Investigation&rft_id=info%3Adoi%2F10.1172%2FJCI58813&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Spirochete+antigens+persist+near+cartilage+after+murine+Lyme+borreliosis+therapy&rft.issn=0021-9738&rft.date=2012&rft.volume=122&rft.issue=7&rft.spage=2652&rft.epage=2660&rft.artnum=http%3A%2F%2Fwww.jci.org%2Farticles%2Fview%2F58813&rft.au=Bockenstedt%2C+L.&rft.au=Gonzalez%2C+D.&rft.au=Haberman%2C+A.&rft.au=Belperron%2C+A.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology">Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy <span style="font-style: italic;">Journal of Clinical Investigation, 122</span> (7), 2652-2660 DOI: <a href="http://dx.doi.org/10.1172/JCI58813" rev="review">10.1172/JCI58813</a></span><br />
<span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.jtitle=Journal+of+Clinical+Investigation&rft_id=info%3Adoi%2F10.1172%2FJCI58813&rfr_id=info%3Asid%2Fresearchblogging.org&rft.atitle=Spirochete+antigens+persist+near+cartilage+after+murine+Lyme+borreliosis+therapy&rft.issn=0021-9738&rft.date=2012&rft.volume=122&rft.issue=7&rft.spage=2652&rft.epage=2660&rft.artnum=http%3A%2F%2Fwww.jci.org%2Farticles%2Fview%2F58813&rft.au=Bockenstedt%2C+L.&rft.au=Gonzalez%2C+D.&rft.au=Haberman%2C+A.&rft.au=Belperron%2C+A.&rfe_dat=bpr3.included=1;bpr3.tags=Biology%2CMicrobiology"> </span>
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<b>Helpful references</b><br />
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Bolz DD, Sundsbak RS, Ma Y, Akira S, Kirschning CJ, Zachary JF, Weis JH, and Weis JJ (August 1, 2004). MyD88 plays a unique role in host defense but not arthritis development in Lyme disease. <cite>The Journal of Immunology</cite> 173(3):2003-2010. <a href="http://www.jimmunol.org/content/173/3/2003.long" target="_blank">Link</a> <br />
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Strle K, Shin JJ, Glickstein LJ, and Steere AC (May 2012). Association of a Toll-like Receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis. <cite>Arthritis and Rheumatism</cite> 64(5):1497-1507. DOI: <a href="http://dx.doi.org/10.1002/art.34383" target="_blank">10.1002/art.34383</a><br />
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Bakaletz LO (October 2007). Bacterial biofilms in otitis media, evidence and relevance. <cite>The Pediatric Infectious Disease Journal</cite> 26(10):S17-S19. <a href="http://www.ncbi.nlm.nih.gov/pubmed/18049376" target="_blank">Link</a><br />
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Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC (December 1999). Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment-resistant Lyme arthritis. <cite>Arthritis and Rheumatism</cite> 42(12):2705-2709. DOI: <a href="http://dx.doi.org/10.1002/1529-0131(199912)42:12%3C2705::AID-ANR29%3E3.0.CO;2-H" target="_blank">10.1002/1529-0131(199912)42:12<2705::aid-anr29>3.0.CO;2-H<!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--><!--2705::aid-anr29--></2705::aid-anr29></a><br />
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<br />
<b>Related posts</b><br />
<ul>
<li><a href="http://spirochetesunwound.blogspot.com/2009/01/chronic-lyme-disease-in-mice.html" target="_blank">Chronic Lyme disease in mice?</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2010/03/tigecycline-fails-to-eradicate.html" target="_blank">Tigecycline fails to eradicate persisting Borrelia burgdorferi</a></li>
<li><a href="http://spirochetesunwound.blogspot.com/2012/02/magic-of-antibiotic-tolerance.html" target="_blank">The magic of antibiotic tolerance</a></li>
</ul>
Microbe Fanhttp://www.blogger.com/profile/00832169199776258021noreply@blogger.com7